Lipid-polymer compounds, compositions, and uses thereof

Abstract

Described herein are compounds comprising lipids attached to a functionalized polymer backbone. Also described herein are uses of such reagents for delivering nucleic acids to cells.

Description

[Technical Field]

[0001] cross reference 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 technology]

[0002] The control of biological processes can be achieved through nucleic acids. Nucleic acids can encode proteins, which, as enzymes, hormones, and other regulatory factors, can carry out processes that allow the organism to function. Nucleic acids can also encode regulatory sequences that control the expression of proteins. Because of their central role in the body, nucleic acids can be ideal therapeutic targets.

[0003] One of the limiting factors for nucleic acid-based therapies is the ability to deliver nucleic acids to the appropriate compartments of cells. Nucleic acids are fragile molecules that can be highly negatively charged (one negative charge per phosphate group) and can be easily cleaved by nucleases present in both extracellular fluids and intracellular compartments. While several attempts to encapsulate or stabilize nucleic acids using proteins, peptides, polymers, lipids, liposomes, and lipid nanoparticles have shown some success, there is a need to identify safe, non-toxic means for stabilizing nucleic acid molecules in biological systems, for example, to enhance the therapeutic efficacy of such therapies. One strategy to achieve this goal is the development of lipid-polymer conjugates that can, for example, interact with (e.g., stabilize) nucleic acid molecules, form liposomes or lipid nanoparticles, and / or penetrate lipid membranes, serving as reporters or functional handles within cell membranes, for example. Additional compositions and methods related to lipid-polymer compounds are described herein. DISCLOSURE OF THE INVENTION

[0004] Summary of the Invention The present disclosure provides lipid-polymer compounds (alternatively "lipid-polymer conjugates") that may be useful for a variety of applications, such as the delivery of biologically active compounds (e.g., nucleic acid molecules) into cells in biological systems, such as in in vitro cell transfection studies. Lipid-polymer conjugates may include one or more lipid compounds attached to a polymer backbone. The present disclosure also provides methods for making such compounds, which may have a variety of applications, such as for use in the treatment of disease or in gene therapy applications.

[0005] In certain aspects, described herein are compounds that include a lipid; and a stimulus-responsive unit. Further examples of lipid and stimulus-responsive unit elements are described below.

[0006] In some aspects, described herein are compounds comprising a lipid; a linker comprising a stimuli-responsive unit; and a polymer, wherein the linker connects the lipid to the backbone of the polymer. In some embodiments, the polymer can comprise, for example, at least three monomer units, wherein the at least three monomer units are C 1-20The stimuli-responsive unit comprises a heteroalkyl side chain. In 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 has a lower critical solution temperature (LCST) of about 27°C to about 35°C. In some embodiments, the temperature-responsive unit has a lower critical 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(Nn-propylacrylamide), poly(N-methyl-Nn-propylacrylamide), poly(N,N-diethylacrylamide), poly(N-isobutylacrylamide), poly(N-sec-butylacrylamide), poly(Nn-butylacrylamide), poly(N-isobutylacrylamide), hydroxypropyl cellulose, 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 to 250 monomer units. More examples of lipids and polymers are described below.

[0007] In one aspect, described herein are compounds comprising a lipid attached to a polymer backbone. The polymer can comprise, for example, at least three monomer units, where the at least three monomer units are C 1-20The polymer may include a heteroalkyl side chain. In some cases, the polymer may include 4 or more (e.g., 10 or more, 50 or more) monomer units. The polymer may include about 400 or less (e.g., about 300 or less) monomer units. In some cases, the polymer may include about 10 to about 200 monomer units. In particular examples, the polymer may include about 50 to about 150 monomer units. The polymer may include a polyacrylate or polyacrylamide. The polymer may include one or more acrylate side chains and / or one or more acrylamide side chains. In some embodiments, each monomer includes an acrylamide or acrylate. The polymer may be a peptide or a non-peptide. As used herein, polymers are generally non-peptides. For example, the polymer may not include an amino acid. In some embodiments, each monomer unit is not an amino acid. The polymer may also include a copolymer. For example, the polymer may include a block copolymer. In some cases, the block copolymer includes cationic or cation-forming monomer units. The block copolymer may be a random block copolymer. In some cases, the polymer is positively charged in neutral aqueous solution. The polymer has a pK of about 2 to about 12 (e.g., about 4 to about 11). b More examples of lipids and polymers are described below.

[0008] In another aspect, provided herein is a compound according to Formula I: XYZ Formula I or a pharmaceutically acceptable salt thereof; X is a lipid; Y is a polymer comprising three or more monomeric units, wherein each of said monomeric units is selected from the group consisting of C 1-20 containing a heteroalkyl side chain; and Z is an unsubstituted or substituted functional group; Here, the lipid is covalently attached to the polymer via the polymer backbone.

[0009] 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 are compounds (e.g., of Formula I) wherein the lipid (X) has a structure of Formula XA, Formula XB, or Formula XC: [ka]

[0010] In some embodiments, provided herein are compounds (e.g., of Formula I) wherein the polymer (Y) has a structure of Formula YA, Formula YB, Formula YC, or Formula YD: [ka] or a pharmaceutically acceptable salt thereof; A 1 , B 1 , C 1 , and D 1 each is independently hydrogen or methyl; A 2 , B 2 , C 2 , and D 2 each independently represents an unsubstituted or substituted C 1-20 is heteroalkyl; each of a, b, c, d, e, and f independently represents an integer from 0 to 200, provided that the total number of monomer units is 3 or more; where substitution C 1-20Each heteroalkyl 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.

[0011] 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 In some embodiments, each of the monomer units independently comprises an acrylate or an acrylamide. [ka] or a pharmaceutically acceptable salt thereof; R 1 and R 3 each is independently hydrogen or methyl; Each R 2 is hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl, C 1-20 heteroalkyl, or a polyethylene glycol chain containing 1 to 100 ethylene glycol monomers; 1-6 Alkyl, C 7-20 Aralkyl, and C 1-20 Each heteroalkyl is unsubstituted or substituted with one or more groups, where each of the one or more groups is independently selected from the group consisting of 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; R4 , and R 5 each independently represents hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl, or C 1-20 heteroalkyl; wherein the C 1-6 Alkyl, C 7-20 Aralkyl, and C 1-20 Each heteroalkyl is unsubstituted or substituted with one or more groups, where each of the one or more groups is independently selected from the group consisting of 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.

[0012] In some embodiments, each of the monomer units independently comprises: [ka]

[0013] 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, a sulfide is an SR 6 and where R 6 is a group of 1 to about 200 atoms selected from hydrogen, halogen, C, N, O, and S. In some embodiments, sulfide is SR 6 and where R 6comprises 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 acceptor. In some embodiments, the capping group is an inert group. In some embodiments, the functional group is selected from the following: [ka] Here, the functional group is attached to the polymer via a sulfur atom.

[0014] In some embodiments, the compound is configured to encapsulate or complex nucleic acids 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) to about 100 kDa.

[0015] In another aspect, described herein are nanoparticles comprising a compound or PLip disclosed herein, the nanoparticles configured for encapsulation or complexation of nucleic acids. In some embodiments, the nanoparticles are configured to encapsulate or complex nucleic acids at a ratio of 0.3:1 to 100:1 (weight:weight). In some embodiments, encapsulation or complexation of nucleic acids increases the half-life of the nucleic acids by at least two-fold under aqueous or physiological conditions. In some embodiments, nuclease digestion of the nucleic acids is inhibited by encapsulation or complexation. In some embodiments, encapsulation or complexation of nucleic acids produces a transfection reagent having an average size of about 20 nm to about 2,000 nm. In some embodiments, complexation includes adsorption of at least a subset of the nucleic acids to the surface of the nanoparticle. In some embodiments, encapsulation or complexation of nucleic acids produces a transfection reagent configured for cellular uptake. In some embodiments, cellular uptake includes endocytosis.

[0016] In another aspect, provided herein are transfection reagents comprising nanoparticles described herein (e.g., comprising a compound disclosed herein) encapsulating genetic information (e.g., one or more nucleic acid molecules). 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 from about 1 kilobase pair (kb) to about 100 kb. In some embodiments, the nucleic acid comprises from about 2 kb to about 20 kb. In some embodiments, the nucleic acid comprises from about 5 kb to about 15 kb. In some embodiments, the nucleic acid comprises from about 8 kb to about 12 kb. In some embodiments, the nucleic acid comprises about 10 kb. In some embodiments, provided herein are transfection reagents having an aqueous solubility of at least 5 μg / mL. In some embodiments, provided herein are transfection reagents having an aqueous solubility of from about 5 μg to about 5 mg / mL. In some embodiments, provided herein are transfection reagents with a water solubility of about 10 μg / mL to about 50 μg / mL.

[0017] In another aspect, provided herein are methods for transfecting cells, the methods 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 contacting is performed 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 a transfection complex. In some embodiments, the conditions sufficient to form a transfection complex comprise conditions sufficient for ionotropic gelation. 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 transfection complex comprises a positive charge under conditions suitable for the entry of the nucleic acid into the cell. In some embodiments, the contact time is less than 24 hours. In some embodiments, the cell comprises an animal cell, a plant cell, a fungal cell, a bacterial cell, or any combination thereof.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising a nanoparticle disclosed herein (e.g., comprising a compound disclosed herein (e.g., of Formula I)) and a biologically active molecule. In some embodiments, the nanoparticle is a lipid nanoparticle. In some embodiments, the nanoparticle is covalently bound to the biologically active molecule. In some embodiments, the nanoparticle is ionically bound to the biologically active molecule. In some embodiments, the nanoparticle encapsulates the biologically active molecule. In some embodiments, the biologically active molecule comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises RNA or DNA. In some embodiments, the nucleic acid molecule comprises mRNA, siRNA, or tRNA. In some embodiments, the biologically active molecule comprises a therapeutic agent. 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.

[0019] 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.

[0020]

[0013] 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 realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications 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 as restrictive. Incorporation by Reference

[0021] All publications, patents, and patent applications mentioned in this specification are herein incorporated 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. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief explanation of the drawings]

[0022] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (herein referred to as "Figure" and "FIG"):

[0023] [Figure 1] FIG. 1 shows the 1H NMR spectrum of the CPCPA-diol of Example S1.

[0024] [Figure 2] FIG. 2 shows the 1H NMR spectrum of the CPCPA-dilinoleyl RAFT agent of Example S2.

[0025] [Figure 3] FIG. 3 shows the 1H NMR spectrum of the CPCPA-cholesterol RAFT agent of Example S3.

[0026] [Figure 4] FIG. 4 shows the 1H NMR spectrum of the cationic diacyl PLip of Example S8(a).

[0027] [Figure 5] FIG. 5 shows the 1H NMR spectrum of P(HPMA)PLip of Example S9.

[0028] [Figure 6] FIG. 6 shows the DNA encapsulation rate of lipid nanoparticles formulated with cationic PLip of Example B1(a).

[0029] [Figure 7] FIG. 7 shows the effect on LNP binding obtained by incorporating 0.5 mol% PLip7 into the LNP formulation of Example B2(b).

[0030] [Figure 8] FIG. 8 shows the dynamic light scattering intensity distribution of LNPs containing PLip6 and 8.

[0031] [Figure 9] FIG. 9 shows anti-spike protein IgG in mice 14 and 35 days after delivery of LNP-encapsulated mRNA, with each bar representing an individual mouse.

[0032] [Figure 10] FIG. 10 shows dose-response priming of Jurkat cells with PLip12 insertion.

[0033] [Figure 11] FIG. 11 shows fluorescence measurements at different temperatures after fluorescent PLip13 was inserted into the plasma membrane of 293F cells.

[0034] [Figure 12] FIG. 12 shows the radius and normalized fluorescence of LNP14-19 after mixing with azidefluor-488.

[0035] [Figure 13] FIG. 13 shows a comparison of 293F cells transfected with cationic PLip20-22 alone.

[0036] [Figure 14] FIG. 14 shows a comparison of 293F cells transfected with TransIT®-Jurkat and POLY1 in the presence or absence of cationic PLip20-22.

[0037] [Figure 15] FIG. 15 shows the effect of temperature cycling on LNP radius for two LNPs containing 0.6 mol % and 1.2 mol % temperature-sensitive PLip23 and one control LNP without PLip23.

[0038] [Figure 16] FIG. 16 shows the effect of temperature cycling on normalized intensity data (particle number) from dynamic light scattering analysis.

[0039] [Figure 17A] FIG. 17A shows the percentage of intact capsids (post-transfection) over time with or without stabilized PLip when assessing complex formation. [Figure 17B] FIG. 17B shows genome numbers (post-transfection) over time with or without stabilized PLip when assessing complex formation.

[0040] [Figure 18A]FIG. 18A shows the complex formation concentration without stabilized PLip as measured by dynamic light scattering (DLS). [Figure 18B] FIG. 18B shows the complex formation concentration in the presence of stabilized PLip as measured by DLS.

[0041] [Figure 19A] FIG. 19A shows the complex formation concentration, genome titer, and percentage of complete capsids measured over time with stabilized PLip. [Figure 19B] FIG. 19B shows another example of complex formation concentration measured over time as genome titer and percent complete capsid with stabilized PLip.

[0042] 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 will be obtained by reference to the following detailed description that sets forth illustrative embodiments. Detailed Description of the Invention

[0043] Regulation of biological processes is mediated by nucleic acids. Nucleic acids encode proteins, which act as enzymes, hormones, and other regulatory factors to carry out processes that enable the body 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 the body.

[0044] A limiting factor in nucleic acid-based therapies is the ability to deliver nucleic acids to the appropriate compartments of cells. Nucleic acids are fragile molecules with a strong negative charge (one negative charge per phosphate group) that are easily cleaved by nucleases present in both extracellular fluids and intracellular compartments. As highly charged molecules, nucleic acids do not cross the lipid membranes surrounding cells and cannot easily escape from endosomal compartments, which are responsible for the uptake of macromolecules into cells. Even RNA interference (RNAi) molecules, despite their small molecular weight, face significant challenges with regard to stability and uptake.

[0045] Efficient delivery of biologically active compounds to the intracellular space of cells has been attempted through the use of a variety of vesicles. Liposomes are microscopic vesicles containing amphiphilic molecules containing both hydrophobic and hydrophilic regions. Furthermore, biologically active compounds can be delivered to the intracellular space of cells via liposome-like structures, such as lipid nanoparticles (LNPs). LNPs can be effective in encapsulating a wide variety of biologically active compounds, such as nucleic acids (mRNA, microRNA, siRNA, etc.). Liposomes and LNPs can also be useful for delivering a wide variety of biologically active compounds, such as nucleic acids, to the intracellular space of cells. This document describes the development, synthesis, and characterization of lipid-polymer compounds and their various applications in treatment and / or detection processes in biological systems.

[0046] While various aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the aspects of the invention described herein may be employed.

[0047] The present invention relates to lipid-polymer compounds (or "lipid-polymer conjugates," or simply "compounds") comprising one or more lipid compounds attached to a polymer backbone that can be used in the delivery of nucleic acids to cells in biological systems, such as, for example, in vitro cell transfection studies. The present invention also relates to methods for making such compounds, and potentially to gene therapy using such compounds.

[0048] The present invention also provides compounds that facilitate the transfer of genetic material (e.g., nucleic acid molecules) into animal cells via complexes comprising nucleic acids and polymers containing ionic or nonionic side chain moieties. 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 combination with endosomolytic lipids to transfect nucleic acids into cells.

[0049] Some of the methods described herein may be useful for altering the expression of one or more genes in a cell or cells.

[0050] Also provided herein are compositions and compounds that, in some cases, can facilitate the delivery of nucleic acids to animal cells (multiple in vitro and / or in vivo).The nucleic acid can comprise a double-stranded structure with a nucleotide sequence substantially identical to a portion of the target nucleic acid expressed in the cell.Furthermore, the use of lipids attached to the backbone of the polymer provided herein can significantly increase the efficiency of nucleic acid transfer.The nucleic acid can then alter the expression of selected endogenous nucleic acids.

[0051] The lipids attached to the polymer backbone described herein can be used to aid in the transfection of DNA, RNA, mRNA, or RNAi into cells, where nucleic acids can alter the natural processes of the cell. definition

[0052] The use of absolute or sequential terms, such as "will," "will not," "shall," "shall not," "must," "must not," "first," "initially," "next," "subsequently," "before," "after," "lastly," and "finally," is not intended to limit the scope of the aspects of the invention disclosed herein, but is used for illustrative purposes.

[0053] As used herein, the singular forms "a," "an," and "the" are generally intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, to the extent that the terms "including," "include," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive, similar to the term "comprising." Similarly, the terms "between," "from," and "to," when referring to ranges, 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-integer numbers, where appropriate) therebetween (e.g., 1 to 4). Similarly, the range "0 to 5" is intended to include 0, 1, 2, 3, 4, and 5.

[0054] Whenever the term "at least," "greater than," or "greater than or equal to" appears before the first number in a series of two or more numbers, the term "at least," "greater than," or "greater than or equal to" applies to each number in that series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0055] Whenever the terms "not greater than," "less than," or "equal to or less than" appear before the first number in a series of two or more numbers, the terms "not greater than," "less than," or "equal to or less than" apply to each number in that series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0056] As used herein, "or" can refer to "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can refer to "A or B," "A but not B," "B but not A," and "A and B." In some cases, context may dictate a particular meaning.

[0057] The term "about" when referring to a numerical value or numerical range generally means that the referenced numerical value or numerical range is an approximation within experimental variability (or statistical experimental error), and that the numerical value or numerical range may vary, for example, by 1% to 15% of the stated numerical value or numerical range. As used herein, the term "about" generally refers to ±10% of the stated numerical value or value. In the case of small numbers (e.g., 1 to 10), the term "about" may refer to ±0.5 of that number. For example, if a variable has a value of "about 2," "about 2" should be understood to refer to a range of 1.5 to 2.5 (inclusive). Similarly, if a variable has a value of "about 4.5," the term should be understood to refer to a range of 4 to 5.

[0058] As used herein, C1-C x is C1-C2, C1-C3, ... , C1-C xをAs an example, a group designated "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 among methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.

[0059] An "alkyl" group generally refers to an aliphatic hydrocarbon group. An alkyl group can be branched or straight-chain. An "alkyl" group is a group having 1 to 10 carbon atoms, i.e., C1-C 10 The term "alkyl" can include alkyl. When a numerical range, such as "1 to 10," is used herein, it refers to each integer in the range; for example, "1 to 10 carbon atoms" means that the alkyl group consists of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 10 carbon atoms, although this definition also covers the use of the term "alkyl" when no numerical range is specified. In some embodiments, alkyl is a C1-C6 alkyl. In one aspect, alkyl is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, or hexyl. The alkyl group can be of any size. In some examples, the alkyl group is a carbon chain about 3 to about 36 carbons long, where 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 alkyl). 30 It may be an alkyl chain or an alkenyl chain.

[0060] An "alkoxy" group may refer to an (alkyl)O- group, where alkyl is as defined herein. In some embodiments, "alkoxy" refers to methoxy (-OCH), ethoxy (-OCHCH), and the like.

[0061] "Hydroxyalkyl" refers to an alkyl in which at least one hydrogen atom has been replaced with hydroxyl. In some embodiments, the hydroxyalkyl is a C1-C4 hydroxyalkyl. Exemplary hydroxyalkyl groups include, but are not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, and the like.

[0062] "Aminoalkyl" refers to an alkyl group in which at least one hydrogen atom is an amine (-NH2, -NHR, -NR2, or -NR3 + In some embodiments, the aminoalkyl is a C1-C6 aminoalkyl. Typical aminoalkyl groups include, but are not limited to, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, and the like.

[0063] The term "alkenyl" may refer to a type of alkyl group in which at least one carbon-carbon double bond is present. In one embodiment, an alkenyl group has the formula -C(H)=CR2, where R refers to the remainder of the alkenyl group, which may be the same or different. In some embodiments, R is H or alkyl. In some embodiments, alkenyl is selected from ethenyl (i.e., vinyl), propenyl (i.e., allyl), butenyl, pentenyl, pentadienyl, and the like. Non-limiting examples of alkenyl groups include -CH=CH-, CH=CH2, -C(CH3)=CH2, -CH=CHCH3, -C(CH3)=CHCH3, and -CH2CH=CH2. Alkenes can be in either the cis (or "Z" configuration) or trans (or "E" configuration). Alkenes can also contain multiple double bonds, each independently E or Z. Preferred are cis alkenes or cis polyalkenes. For example, the cis alkene can be an oleyl group (eg, oleic acid or ester).

[0064] 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, an alkynyl group has the formula -C≡CR, where R refers to the remainder of the alkynyl group. In some embodiments, R is H or alkyl. In some embodiments, alkynyl 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.

[0065] The term "heteroalkyl" generally refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, sulfur, or combinations thereof. The heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. In one aspect, the heteroalkyl is a C1-C6 heteroalkyl. In one aspect, the heteroalkyl is a C6-C 30Heteroalkyl. Heteroalkyl groups can include nitriles, amides, esters, ethers, amines, thioethers, thioesters, carbamates, carbonates, polyethers, polyamines, etc. Heteroalkyl groups can include alkyl ethers (e.g., polyethers), alkyl esters (e.g., polyesters), alkyl amines (e.g., polyamines), alkyl amides (e.g., polyamides), or any combination thereof. The heteroalkyl group can include an acrylate or acrylamide. In some examples, the heteroalkyl group includes an ether or polyethylene glycol (PEG) group, where the PEG group includes 2 to 100 monomer units. As a non-limiting example, each of A1-A12 can be considered to have a heteroalkyl side chain.

[0066] 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 aryl ("aryl", e.g., phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups.

[0067] 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, this term distinguishes carbocycles from "heterocyclic" rings or "heterocycles," in which the ring backbone contains at least one atom other than carbon. In some embodiments, at least one of the two rings in a bicyclic carbocycle is aromatic. In some embodiments, both rings in a bicyclic carbocycle are aromatic. Carbocycles include aryl and cycloalkyl.

[0068] As used herein, the term "aryl" generally refers to an aromatic ring in which each of the atoms 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 carbocycle 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).

[0069] The term "cycloalkyl" may refer to a monocyclic or polycyclic aliphatic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl is optionally fused to an aromatic ring, and the point of attachment is at a carbon that is not an aromatic ring carbon atom. Cycloalkyl groups include groups having 3 to 10 ring atoms. In some embodiments, the cycloalkyl group is selected from among cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, spiro[2.2]pentyl, norbornyl, and bissilyl[1.1.1]pentyl. In some embodiments, the cycloalkyl is a C3-C6 cycloalkyl. In some embodiments, the cycloalkyl is a C3-C4 cycloalkyl.

[0070] The term "halo," or alternatively "halogen" or "halide," generally refers to fluoro, chloro, bromo, or iodo. In some cases, halo may refer to fluoro, chloro, or bromo. As used herein, the term "halo" is not intended to be numerically limiting. For example, a haloalkyl group can contain one, two, three, or more halogen groups. A haloalkyl group can be, for example, -CHF, -CHF, or -CF.

[0071] The term "fluoroalkyl" generally refers to an alkyl in which one or more hydrogen atoms have been replaced with a fluorine atom. In one aspect, the fluoroalkyl is a C1-C6 fluoroalkyl. Examples of fluoroalkyl groups include, but are not limited to, -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CF3, -CF2CF3, and the like.

[0072] The term "heterocycle" or "heterocyclic" generally refers to heteroaromatic (also known as heteroaryl) and heterocycloalkyl rings containing 1 to 4 heteroatoms in the ring(s), where each heteroatom in the ring(s) is selected from O, S, and N, and where each heterocyclic group has 3 to 10 atoms in its ring system, with the proviso that no ring contains two adjacent O or S atoms. Non-aromatic heterocyclic groups (also known as heterocycloalkyl) contain rings having 3 to 10 atoms in the ring system, and aromatic heterocyclic groups contain rings having 5 to 10 atoms in the ring system. Heterocyclic groups include benzo-fused ring systems. Examples of non-aromatic heterocyclic groups include pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithio ranyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, indolin-2-onyl, isoindolin-1-onyl, isoindolin-1,3-dionyl, 3,4-dihydroisoquinolin-1(2H)-onyl, 3,4-dihydroisoquinolin-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 quinolidinyl.Examples of aromatic heterocyclic groups are pyridinyl, 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. The foregoing groups are either C-attached (or C-linked) or N-attached where possible. For example, groups derived from pyrrole include both pyrrol-1-yl (N-linked) and pyrrol-3-yl (C-linked).Further, groups derived from imidazole include imidazol-1-yl or imidazol-3-yl (both N-linked), or imidazol-2-yl, imidazol-4-yl, or imidazol-5-yl (all C-linked).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 a bicyclic heterocycle is aromatic.In some embodiments, both rings of a bicyclic heterocycle are aromatic.

[0073] The term "heteroaryl" or alternatively "heteroaromatic" generally refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. Illustrative examples of heteroaryl groups include, but are not limited to, monocyclic heteroaryls and bicyclic heteroaryls. Monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Monocyclic heteroaryls 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 contains 0-4 N atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl contains 0-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl is a C1-C9 heteroaryl. In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5- or 6-membered heteroaryl.

[0074] A "heterocycloalkyl" group generally refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. In some embodiments, the heterocycloalkyl is fused with an aryl or heteroaryl. In some embodiments, the heterocycloalkyl is oxazolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidin-2-onyl, pyrrolidine-2,5-dithionyl, pyrrolidine-2,5-dionyl, pyrrolidinonyl, imidazolidinyl, imidazolidin-2-onyl, or thiazolidin-2-onyl. In one aspect, the heterocycloalkyl is C2-C 10 In some embodiments, heterocycloalkyl is C-C 10 Heterocycloalkyl. In some embodiments, heterocycloalkyl is monocyclic or bicyclic. In some embodiments, heterocycloalkyl is monocyclic and has a 3-, 4-, 5-, 6-, 7-, or 8-membered ring. In some embodiments, heterocycloalkyl is monocyclic and has a 3-, 4-, 5-, or 6-membered ring. In some embodiments, heterocycloalkyl is monocyclic and has a 3- or 4-membered ring. In some embodiments, heterocycloalkyl contains 0-2 N atoms in the ring. In some embodiments, heterocycloalkyl contains 0-2 N atoms, 0-2 O atoms, and 0-1 S atoms in the ring.

[0075] The term "bond" or "single bond" generally refers to a chemical bond between two atoms or two 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 forming a bond between the remaining specified groups.

[0076] The term "moiety" generally refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized as chemical entities embedded within or appended to a molecule.

[0077] The term "substituted" generally refers to a group in which one or more hydrogen atoms have been replaced with a substituent, for example, a substituent selected from the group consisting of halogen, -CN, -NH, -NH(alkyl), -N(alkyl), -OH, -COH, -COalkyl, -C(=O)NH, -C(=O)NH(alkyl), -C(=O)N(alkyl), -S(=O)NH, -S(=O)NH(alkyl), -S(=O)N(alkyl), alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthiol, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituents are independently selected from halogen, -CN, -NH, -NH(CH), -N(CH), -OH, -COH, -CO(C-C alkyl), -C(=O)NH, -C(=O)NH(C-C alkyl), -C(=O)N(C-C alkyl), -S(=O)NH, -S(=O)NH(C-C alkyl), -S(=O)N(C-C alkyl), C-C alkyl, C-C cycloalkyl, C-C fluoroalkyl, C-C heteroalkyl, C-C alkoxy, C-C fluoroalkoxy, -SC-C alkyl, -S(C-C)C-C alkyl, and -S(=O)C-C alkyl. In some embodiments, optional substituents are independently selected from halogen, -CN, -NH, -OH, -NH(CH), -N(CH), -CH, -CHCH, -CHF, -CF, -OCH, -OCHF, and -OCF. In some embodiments, substituents are substituted with one or two of the preceding groups. In some embodiments, optional substituents on an aliphatic carbon atom (acyclic or cyclic) include oxo (=O). A group may be "optionally substituted," meaning that the group may be unsubstituted or substituted as described above.

[0078] The term "acrylate" generally refers to the salt, ester, or conjugate base of acrylic acid. These acrylates (CH=CHC0R or CH=CHC0H) contain a vinyl group that is susceptible to polymerization, and the carboxylate group has various functional groups. Modified acrylates include, but are not limited to, methacrylates (CH=C(CH)C0R or CH=C(CH)C0H) and cyanoacrylates (CH=C(CN)C0R or CH=C(CN)C0H). As used herein, the term "acrylate" includes such modified acrylates, including "methacrylates." As used herein, the term "acrylate" includes "methacrylates."

[0079] The term "acrylamide" generally refers to vinyl-substituted primary, secondary, or tertiary amides (CH=CHC(O)NH, CH=CHC(O)NHR, or CH=CHC(O)NR1R2). Modified acrylamides include, but are not limited to, methacrylamide (CH=C(CH3)C(O)NH, CH=C(CH3)C(O)NHR, or CH=C(CH3)C(O)NR1R2) and cyanoacrylamide (CH=C(CN)C(O)NH, CH=C(CN)C(O)NHR, or CH=C(CN)C(O)NR1R2). As used herein, the term "acrylamide" includes such modified acrylamides, including "methacrylamide." As used herein, the term "acrylamide" includes "methacrylamide."

[0080] A "polymer" generally refers to a molecule composed of repeated bonds of smaller units called monomers. As used herein, the term "polymer" can include both oligomers, which can have from 2 to about 80 monomers, and polymers having more than 80 monomers. A polymer can contain, for example, 4 or more monomer units (e.g., 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 monomer units or monomers). A polymer can contain, for example, about 1,000 or fewer monomer units or monomers (e.g., about 500 or fewer, about 400 or fewer, about 300 or fewer, about 200 or fewer, about 150 or fewer, about 100 or fewer, or about 50 or fewer monomer units or monomers). In some examples, a 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). Polymers can be linear, branched network, star, comb, or ladder polymers. Polymers can be homopolymers, where a single monomer is used, or copolymers, where two or more monomers are used. Types of copolymers include alternating, random, block, and graft. The "backbone" or "backbone" of a polymer can refer to the longest series of covalently bonded atoms that together form a continuous chain of a particular molecule. The backbone or backbone of a polymer can be composed of atoms with bonds necessary to extend the length of the polymer in a step-growth or chain-growth polymerization. The side chains of a polymer can be composed of atoms with bonds not necessary to extend the length of the polymer. Those skilled in the art of polymerization technology will recognize several categories of polymerization processes that can be utilized in the described processes. In some embodiments, the polymers described herein have heteroalkyl side chains (e.g., acrylate or acrylamide side chains). The polymer can include an alkyl backbone. The polymer can include an alkenyl backbone. The polymer can include a heteroalkyl backbone. Examples of heteroalkyl backbones include, but are not limited to, polyethers (eg, polyethylene glycol, or PEG) and polyamines (eg, polyethyleneimine, or PEI).

[0081] "Monomer" generally refers to a building block that constructs a polymer. As used herein, a "monomer" can be a divalent chemical unit, where each valency of the unit is completed by bonding to an adjacent monomer (thereby producing a polymer) (e.g., via polymerization) or by bonding to an end group (e.g., a lipid or functional group as described herein). The monomers described herein can be bonded to adjacent monomers or end groups via a backbone. Monomers can be bonded to adjacent monomers or end groups via a bond or by a linker (e.g., C optionally substituted with an amine, amide, ether, ester, carbonyl, carbamate, carbonate, etc.). 1-20 The polymer may be linked to an adjacent group (e.g., a second monomer, a lipid, or a functional group) via a linker (e.g., an alkyl linker). For example, the polymer may be linked to a lipid or functional group via a linker, where the linker is C 1-20 Alkyl esters (e.g., C 1-6 alkyl esters).

[0082] "Side chain" or "side-chain" generally refers to a group of atoms in a monomer attached to the backbone, which does not contribute to chain elongation and may optionally have additional functionality. For example, a side chain having an amino group (e.g., linear, branched, cyclic, or primary, secondary, tertiary, or quaternary amine) capable of forming a cation. In some embodiments, a side chain with basic or cationic properties interacts with the acidic or anionic phosphate backbone of a nucleic acid molecule, thereby enhancing the ability of the monomer (or a compound containing such a monomer) to bind to nucleic acid. In some embodiments, a cationic or basic side chain interacts with the anionic phosphate group of a nucleic acid molecule, thereby forming a net-neutral (or more neutral than unbound nucleic acid) molecule that can then cross a cell membrane. In some embodiments, the side chain is specifically designed to facilitate transport of charged molecules (e.g., nucleic acid molecules) into or out of cells. In some embodiments, the side chain is not cationic. In some embodiments, the side chains are heteroalkyl groups that modulate 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 can have no side chains (e.g., PEG or PEI groups). In some embodiments, a polymer comprises repeating units of two or more monomers, thereby forming an ABABAB pattern of side chains, where A and B represent the same or different side chains.

[0083] The term "biologically active compound" or "bioactive compound" generally refers to a chemical compound that has known or suspected biological activity in a mammal. As used in this context, a chemical 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, and nucleic acid molecules (e.g., DNA or RNA). Included in the term "biologically active molecule" are all cations, anions, salts, oxides, solvates, stereoisomers, and isotopes that have 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 containing radioisotopes), drugs (e.g., antiproliferative agents, antineoplastic agents, cytotoxic or cytostatic agents, targeted therapeutic agents, anti-inflammatory agents, nucleic acid molecules, peptides, etc.). As disclosed herein, some examples of bioactive 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 compound or bioactive compound may refer to payloads (e.g., therapeutic payloads) disclosed herein.

[0084] As used herein, the term "payload" generally refers to a molecule that has utility within a target cell. A payload may be useful in that it can be detected or manipulated within the cell. A payload can also be a biologically active or bioactive molecule. In such cases, the payload may exert a biological effect (e.g., a disease-modifying effect) within the target cell. Nucleic acid molecules such as plasmid DNA, mRNA, and siRNA may be particularly useful as payloads. Lipid nanoparticles comprising a compound disclosed herein and a nucleic acid molecule payload may be particularly useful as transfection reagents, i.e., reagents for introducing nucleic acids into eukaryotic cells. A payload may also include several different classes of molecules. For example, a payload may refer to a combination of a nucleic acid molecule and one or more additional non-nucleic acid molecules (e.g., small molecule therapeutics, chelators, binders, etc.). A payload may also include a detectable agent or detectable group (e.g., a metal, a radioactive tracer, a dye, etc.), a therapeutic agent (e.g., an immunomodulator, an anticancer agent, an antiviral agent, etc.), an oligonucleotide (e.g., siRNA, mRNA), etc.

[0085] "Therapeutic agent," or "therapeutic agent," or "therapeutic payload" generally refers to a disease-modifying or pathogen-directed agent. Therapeutic agents include any drug that is 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, inotersen, patisiran, voranesorsen, givosiran, golodirsen, viltolarsen, lumasiran, inclisiran, and casimersen are examples of therapeutic agents that can be used in combination with the LNPs and PLips disclosed herein.

[0086] As used herein, "nucleic acid" or "nucleic acid molecule" generally refers to any biopolymer containing nucleotides (e.g., cytosine, guanine, adenine, uracil, or thymine). In some embodiments, a nucleic acid comprises naturally occurring nucleotides. A nucleic acid can comprise non-naturally occurring nucleotides. In some embodiments, a nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or any combination thereof. In some embodiments, a nucleic acid comprises deoxyribonucleic acid (DNA). In some embodiments, a nucleic acid comprises ribonucleic acid (RNA). In some embodiments, a nucleic acid comprises locked nucleic acid (LNA). In some embodiments, a nucleic acid comprises peptide nucleic acid (PNA). In some embodiments, a DNA is single-stranded DNA (ssDNA). In some embodiments, a DNA is double-stranded DNA (dsDNA). In some embodiments, a DNA is circular DNA (cDNA), e.g., plasmid DNA. In some embodiments, a DNA is recombinant DNA (rDNA). In some embodiments, a DNA is genomic DNA. In some embodiments, a DNA is synthetic, modified, or non-naturally occurring DNA. In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the RNA is transfer RNA (tRNA). In some embodiments, the RNA is ribosomal RNA (rRNA). In some embodiments, the RNA is small nuclear RNA (snRNA). In some embodiments, the RNA is microRNA (miRNA). In some embodiments, the RNA is silencing RNA (siRNA). In some embodiments, the RNA is naked RNA (e.g., unenveloped RNA that is not complexed with lipids, proteins, or other stabilizing or protective molecules). In some embodiments, the RNA is complexed RNA.

[0087] As used herein, an "effective amount" or "therapeutically effective amount" generally refers to a sufficient amount of an administered drug or compound (e.g., a payload or biologically active molecule) to alleviate to some extent one or more of the symptoms of the disease or condition being treated. This result includes reduction or alleviation of the signs, symptoms, or causes of a disease, or other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound disclosed herein required to provide a clinically significant reduction in a disease symptom. An appropriate "effective" amount in any individual case is optionally determined using techniques such as dose escalation studies.

[0088] "Dispersity" or "polydispersity index" generally refers to a measure of the breadth of a polymer's molecular weight distribution, which is M w / M n where M w is the weight-average molecular weight (or "mass-average molar mass"), and M n is the number average molecular weight (or "number average molar mass"). This value corresponds to the heterogeneity in size of the molecules in a mixture. For a mixture where all the molecules are the same size, the dispersity is 1. A lower dispersity may indicate greater uniformity within the sample.

[0089] "Steric stabilizers" generally refer to long-chain hydrophilic groups that sterically hinder particle-particle electrostatic interactions, thereby preventing aggregation of the final polymer. Examples include, but are not limited to, alkyl groups, PEG chains, polysaccharides, and alkylamines. Electrostatic interactions are non-covalent bonds between two or more substances due to the attractive forces between positive and negative charges.

[0090] A "reactive group" generally refers to a chemical moiety that can form either an ionic or covalent bond with another compound, particularly in a biological context (e.g., at about neutral pH and about 37°C). The portion of a reactive compound that can form a covalent bond is sometimes referred to as a reactive functional group. Reactive groups include binding partners for specific reactions (e.g., alkynes or azides for "click" type chemistry or reactions), as well as generally reactive species (e.g., nucleophiles or electrophiles). In some embodiments, the reactive group is a maleimide or succinimide. In some embodiments, the reactive group is a chemical group capable of performing a bioorthogonal reaction. In some embodiments, the bioorthogonal 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 monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dibenzoazacyclooctyne (DIBAC), biarylazacyclooctyne (BARAC), dimethoxyazacyclooctyne (DIMAC), and the like.

[0091] A "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 comprises a conjugated aromatic or heteroaromatic system that can emit light in response to a stimulus (e.g., excitation). In some embodiments, the detectable group comprises 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 comprises a pyrene group, a benzopyrene group, or a derivative thereof. Fluorescent groups, alternatively "fluorophores," are well known in the art, and all known fluorophores are considered within the scope of the present invention. In some embodiments, the fluorophore is a fluorescein, rhodamine, coumarin, cyanine, xanthene, or a derivative thereof. In some embodiments, the fluorophore is an Alexa Fluor or DyLight Fluor probe, or a derivative thereof.

[0092] "Steroid" or "steroid derivative" generally refers to a sterol or stanol, or a steroid hormone, or analog thereof, in which a hydroxyl moiety has been modified (e.g., acylated). Modifications may include spacer groups, linkers, reactive groups. As used herein, a steroid can be a naturally occurring or non-naturally occurring steroid known in the art (e.g., cholesterol), or a derivative thereof.

[0093] 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.

[0094] As used herein, the term "in vivo" may be used to describe events that occur within an organism, such as the body of a subject. In some embodiments, in vivo refers to events that occur within the body of a non-human subject, such as a mouse or rat. In some embodiments, in vivo refers to events that occur within the human body.

[0095] As used herein, the term "in vitro" may be used to describe events that occur in a container that holds an experimental reagent and is separated from the living source organism from which the material was obtained. In vitro assays may include cell-based assays that use live or dead cells. In vitro assays may also include cell-free assays that do not use intact cells. Lipid-Polymer Compounds (“PLips” or alternatively “Compounds”)

[0096] In one aspect, disclosed herein is a polymer-lipid compound comprising a lipid, a polymer, and a functional group. The lipid may be attached to the polymer via the backbone. The lipid may also be attached to the polymer backbone via a (non-side chain linker). The polymer may comprise a backbone and side chains (e.g., C 1-20 The polymer may contain heteroalkyl side chains (heteroalkyl side chains). Alternatively, the polymer may have no side chains. In some cases, polymers containing heteroalkyl side chains interact with nucleic acid molecule(s), thereby stabilizing or encapsulating the nucleic acid molecule(s). Polymers containing heteroalkyl side chains may provide advantageous biological or physicochemical properties compared to unsubstituted (e.g., PEG or PEI) polymers.

[0097] In another aspect, disclosed herein is a compound having a structure according to Formula I: XYZ or a pharmaceutically acceptable salt thereof; X is a lipid; Y is a polymer comprising at least three monomeric units, each of said monomeric units being selected from the group consisting of C 1-20 containing a heteroalkyl side chain; and Z is an unsubstituted or substituted functional group; Here, the lipid is covalently attached to the polymer via the backbone. Lipid (“X”)

[0098] The "lipid" component(s) of the polymer-lipid compounds described herein generally refer to an organic compound (or, in the case of a polymer-lipid compound, a radical thereof) that is or contains a lipophilic and / or hydrophobic moiety. Lipids often have low water solubility but are soluble in non-polar solvents. As used herein, the term "lipid" may refer to either the lipophilic group itself or a derivative thereof. The lipid may further comprise a linker or spacer. For example, the lipid may comprise a hydrophobic moiety with an optionally substituted alkyl or optionally substituted heteroalkyl linker connecting the hydrophobic moiety to the polymer (Y). In some cases, the lipid is 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 (mono)acyl, diacyl, or triacylglycerides. As described herein, the lipid may be (or may include) an acylglyceride or diacylglyceride attached to a polymer backbone.

[0099] Lipids may also refer to non-polar groups that do not contain fatty acids. For example, sterols, phospholipids, or their derivatives may be referred to as lipids. The lipids described herein may include a linker that connects monoglycerides, diglycerides, sterols, etc. to the polymer backbone. The linker may be a C 1-6 It may be an alkyl group or a 1- to 6-membered heteroalkyl group, each of which is optionally further substituted. In some cases, the linker is a substituted or unsubstituted C 1-6The linker group is an alkyl group, which forms an ester bond with the hydroxy group of glycerol or sterol. When substituted, the linker group may be substituted with, for example, 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 may be substituted or unsubstituted C 1-6 The linker is also an alkyl ester. 1-6 Ether, C 1-6 Amide, C 1-6 Carbamate, C 1-6 Phosphoester, C 1-6 Alternatively, longer linkers containing from about 7 to about 20 carbon atoms, attached via an ester bond and optionally substituted as described above, can be employed.

[0100] As used herein, lipids may refer to any steroid (e.g., sterol). For example, in some instances, lipids include sterols or stanols. Examples of sterols include, but are not limited to, cholesterol, campesterol, stigmasterol, brassicasterol, avenosterol, sitosterol, and ergosterol.

[0101] Lipids can include one or more fatty acids. For example, lipids can be diacylglycerides containing one or more saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, or combinations thereof. Fatty acids generally contain an acid group and a hydrocarbon chain (alternatively, a fatty acid tail). The fatty acid tail can range in length from about 10 to about 30 carbon atoms and have a degree of unsaturation between about 0 and about 6 degrees. As used herein, a fatty acid tail refers to a fatty acid having a C 10-30 Alkyl group, C 10-30 Alkenyl group, or C 10-30 The fatty acid tail may be an alkynyl group. The fatty acid tail may be substituted or unsubstituted. In some cases, the fatty acid tail may be an unsubstituted C 10-30 Alkyl group or C 10-30In some cases, the fatty acid tail is a straight or unbranched C alkenyl group. 10-30 Alkyl group or C 10-30 It is an alkenyl group.

[0102] In one example, the lipid comprises a monoacylglycerol (MAG) or diacylglycerol (DAG) group with one or more linoleic or oleic acid tail(s). The lipid further comprises a geminal disubstituted C linking the MAG or DAG to the polymer backbone. 1-6 In another example, the lipid comprises a cholesterol group. The cholesterol lipid may further comprise a geminal disubstituted C linker linking the sterol to the polymer backbone. 1-6 It may also include an alkyl ester linker.

[0103] As an example, the lipid may have a structure represented by any one of the following formulas: [ka] where each L is a substituted or unsubstituted C 1-6 is an alkyl group, and S 1 is the sterol group, and FA 1 , F.A. 2 , F.A. 3 , and F.A. 4 Each of L is a fatty acid 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 The linker L is an alkyl group or a substituted or unsubstituted 2-12 membered heteroalkyl group. As described herein, L can include geminal substitutions (e.g., dimethyl or methyl / cyano). In other examples, the linker L is monosubstituted. In still other cases, the linker L is unsubstituted. Examples of linkers include, but are not limited to, the following: [ka]

[0104] In further examples, the lipid may have a structure represented by any one of the following formulas: [ka]

[0105] As used herein, alkenyl includes dienes and trienes and can include both cis (or E) and trans (or Z) isomers. All forms of stereoisomers (e.g., enantiomers, diastereomers, etc.) are considered within the scope of the above formula throughout this disclosure unless otherwise specified.

[0106] The lipid may be hydrophobic. In some instances, the lipid is amphiphilic. In some embodiments, the lipid has 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 has a log(K OW In a further example, the lipid has a log(K OW ) is in the 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. The lipids may have a log(K) in the range of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10. OW The lipid may have a log(K) of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, or about 9. 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 ).

[0107] The compounds disclosed herein can have a lipid having the following formula: [ka] Here, FA 1 and F.A. 2 are each independently an ester side chain derived from one of the following fatty acids: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0108] In further examples, the compounds disclosed herein can have a lipid of the following formula: [ka] Here, FA 1 and F.A. 2 Each of the 1-30 Alkyl, C 1-30 Alkenyl, C 1-30 alkynyl, or 1-30 membered heteroalkyl. [ka]

[0109] The compounds disclosed herein can have a lipid of the following formula: [ka] Polymer ("Y")

[0110] PEG-DMG is a widely used stabilizing lipid in LNPs. Several studies and clinical reports suggest that moderate to severe immunogenic reactions can occur after systemic administration of PEG. Acute hypersensitivity and reduced therapeutic efficacy of PEGylated drugs may be due to activation of the complement system and / or anti-PEG antibody production. Various stabilized PLips have been synthesized and shown to form stable LNPs of comparable size compared to LNPs stabilized with PEG-DMG. Stabilized PLips contain any variation of lipid tails (cholesterol, diacyl, acyl, etc.), while the polymer side chain(s) can contain hydrophilic stabilizing groups. The stabilizing polymer side chains are sometimes referred to as heteroalkyl side chains, stabilizing side chains, noncationic side chains, or solubilizing side chains. Some examples of stabilizing 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 LNPs can depend on the nature and length of the polymer side chains. Furthermore, the size and stability of LNPs can be determined or influenced by the identity of the lipid tail present in the stabilized PLip. In some cases, stabilized PLips are used as an alternative to PEG stabilizers (e.g., PEG-DMG). Alternatively, PLips can have both stabilizing and cationic properties. For example, PLips can contain multiple polymer blocks (sequential, alternating, statistical, random), where one block (or multiple identical monomer units) contains cationic or cation-forming groups, while the second block (or multiple identical monomer units) contains stabilizing side chains. Stabilizing PLips can include polymers with alkoxy or hydroxy side chains. In some cases, stabilized PLips are hydrophilic. Stabilizing PLips can contain from about 3 to about 200 stabilizing monomer units. The cationic monomer units can have a structure according to any of the following formulas: [ka] where each R group is C 1-6 Alkyl, C 1-6 Alkoxyalkyl, C 1-6 hydroxyalkyl, or R is a polyethylene glycol (PEG) group containing 1 to 100 PEG units; and each R' group is hydrogen or methyl.

[0111] Some examples of stabilizing monomer units include, but are not limited to, the following: [ka] where each R' is hydrogen or methyl. More specifically, the stabilizing monomer unit can have a structure according to any of the following formulas: [ka]

[0112] Cationic PLips can contain any variation of lipid tails (cholesterol, diacyl, acyl, etc.), while the polymer side chain(s) can contain cationic or cation-forming groups (e.g., amines). For example, cationic PLips can include cationic polymers, where at least one (e.g., at least three) monomer units of the polymer contain cationic or cation-forming side chains. As used herein, cationic is intended to include not only cations but also uncharged groups that become cationic in a biological environment. For example, the cationic side chains can include amide or ester groups, where the amide or ester is substituted with an alkylamine or alkylammonium side chain. Cationic PLips can contain from about 3 to about 200 cationic monomer units. The cationic monomer units can have a structure according to any of the following formulas: [ka] wherein each R group is an alkylamine or alkylammonium, and wherein each R' group is hydrogen or methyl. More specifically, the cationic monomer unit has a structure of any of the following formulas: [ka] or a salt thereof, wherein each R' is independently hydrogen or methyl.

[0113] Some examples of cationic monomer units include, but are not limited to, the following: [ka] In each of the above monomer unit examples, the alkyl chain can be 2 to 6 carbons in length (e.g., 2, 3, or 4 carbons in length). For example, in any one of the above monomer units, the alkyl chain can be an ethyl chain, a propyl chain, or a butyl chain. In certain embodiments, the cationic monomer unit is selected from the group consisting of: [ka] or a salt thereof, or the free base thereof.

[0114] In some examples, a polymer (e.g., a polymer having a structure represented by formula YA, formula YB, formula YC, or formula YD) includes at least a first plurality of monomer units represented by any of the following formulas: [ka] Here, a is an integer of 3 to 400, and n is an integer of 1 to 100.

[0115] 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 specific examples disclosed herein, n has an average value of 2 to 10, 3 to 6, or 4 to 5 (e.g., n is about 4.5). n can be an integer having an average value of about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0116] In another aspect, provided herein are compounds comprising a lipid attached to a polymeric backbone, wherein the polymer comprises three or more monomeric units. In some cases, the monomeric units are C 1-20 Contains heteroalkyl side chains.

[0117] The polymer may contain about 5 or more monomer units, about 10 or more monomer units, about 15 or more monomer units, about 20 or more monomer units, about 30 or more monomer units, about 50 or more monomer units, about 100 or more monomer units, about 200 or more monomer units, about 300 or more monomer units, or about 400 or more monomer units. Each monomer unit may have a C 1-20 It may also contain heteroalkyl side chains.

[0118] In some cases, the polymer can include from about 3 monomer units to about 400 monomer units. For example, the polymer can include 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, about 4 monomer units to about 50 monomer units, 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 to about 150 monomer units, about 10 to about 200 monomer units, about 10 to about 400 monomer units, about 30 to about 50 monomer units, about 30 to about 80 monomer units, about 30 to about 100 monomer units, about 30 to about 120 monomer units, about 30 to about 150 monomer units, about 30 to about 200 monomer units, about 30 to about 400 monomer units, about 50 to about 80 monomer units - 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,The polymer may comprise about 100 to about 150 monomer units, about 100 to about 200 monomer units, about 100 to about 400 monomer units, about 120 to about 150 monomer units, about 120 to about 200 monomer units, about 120 to about 400 monomer units, about 150 to about 200 monomer units, about 150 to about 400 monomer units, or about 200 to about 400 monomer units. In some embodiments, the polymer comprises about 3 monomer units, 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. In some embodiments, the polymer comprises at least about 3, 4, 10, 30, 50, 80, 100, 120, 150, or 200 monomer units. In some embodiments, the polymer comprises at most about 4, 10, 30, 50, 80, 100, 120, 150, 200, or 400 monomer units. Each monomer unit is represented by C, 1-20 Contains heteroalkyl side chains.

[0119] The polymer may be a polyacrylate (also called a poly(acrylic acid ester) or a poly(acrylic acid alkyl ester)). A polyacrylate polymer has one or more monomer units comprising an acrylate (also called an acrylic acid ester or an acrylic acid alkyl ester). A polyacrylate polymer may have about 10 or more monomer units comprising an acrylate. In some cases, the polymer may have up to about 400 monomer units comprising an acrylate. For example, the polymer may have about 10 to about 400 monomer units comprising an acrylate. In other examples, the polymer may have about 20 to about 300, about 50 to about 200, or about 100 to about 150.

[0120] In some embodiments, the polymer is polyacrylamide. In some embodiments, the polymer has monomer units that include acrylamide (also called acrylic amide). In some embodiments, the polymer includes a combination of acrylate and acrylamide.

[0121] In some embodiments, the polymer comprises a series of repeat units, wherein the repeat units comprise two, three, or four monomers. In some embodiments, the repeat units comprise both acrylamide and acrylate. In some embodiments, the repeat units comprise two or more acrylamides. In some embodiments, the repeat units comprise two or more acrylates.

[0122] In some embodiments, the polymer is an acrylate-containing homopolymer. In some embodiments, the homopolymer comprises cationic monomer units. In some embodiments, the homopolymer is positively charged in neutral aqueous solution.

[0123] 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.

[0124] In some embodiments, the polymer is a copolymer, including 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 neutral aqueous solution.

[0125] In some embodiments, the polymer is a cationic polymer, e.g., comprising multiple cationic or cation-forming groups (e.g., primary amines). In some embodiments, the cationic polymer comprises multiple monomer units, where each monomer unit comprises an aminoalkyl side chain (e.g., CH2CH2NH2, CH2CH2CH2NH2, or a cation thereof). In some embodiments, the cationic polymer comprises multiple monomer units, where each monomer unit comprises an alkylammonium side chain (e.g., CH2CH2N(CH3)3). + , CH2CH2CH2N(CH3)3 + In some embodiments, the cationic PLip comprises at least a first plurality of monomeric units, wherein each said monomeric unit of said first plurality of monomeric units comprises an aminoalkyl (or alkylamino) side chain (e.g., —NH or —N(CH) + In some embodiments, the cationic PLip comprises a second plurality of monomeric units, wherein each said monomeric unit of said second plurality of monomeric units comprises a hydroxyalkyl or alkoxyalkyl side chain.

[0126] In some embodiments, the polymer has a pK in the range of about 2 to about 12. bIn some embodiments, the polymer has 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 pK in the range of 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5 to about 11, about 5 to about 12, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 11, about 6 to about 12, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 11, about 7 to about 12, about 8 to about 9, about 8 to about 10, about 8 to about 11, about 8 to about 12, about 9 to about 10, about 9 to about 11, about 9 to about 12, about 10 to about 11, about 10 to about 12, or about 11 to about 12 b In some embodiments, the polymer has 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 has 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 has a pK in the range of up to about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12. b It has.

[0127] The polymer may have a structure represented by formula YA, formula YB, formula YC, or formula YD: [ka] or a pharmaceutically acceptable salt thereof; A 1 , B 1 , C 1 and D 1 is independently selected at each instance from hydrogen and methyl; A 2 , B 2 , C 2 and D 2is, independently in each instance, C 1-20 heteroalkyl; a, b, c, d, e, and f are, in each instance, independently an integer selected from 0 to 400 (e.g., 0 to 200 (e.g., 40 to 120)), provided that the total number of monomer units is 3 or more; Here, C 1-20 When heteroalkyl is substituted, it is substituted with unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl.

[0128] In some embodiments, cycloalkyl is C 3-24 In some embodiments, the cycloalkyl is a C 3-15 In some embodiments, the cycloalkyl is a C 3-10 In some embodiments, the cycloalkyl is a C 3-6 It is a cycloalkyl ring. In some embodiments, the heterocyclyl is a 3- to 24-membered heterocycle. In some embodiments, the heterocyclyl is a 3- to 15-membered heterocycle. In some embodiments, the heterocyclyl is a 3- to 10-membered heterocycle. In some embodiments, the heterocyclyl is a 3- to 6-membered heterocycle. In some embodiments, the heterocyclyl is oxirane, oxetane, tetrahydrofuran, tetrahydropyran, aziridine, azetidine, pyrrolidine, piperidine, piperazine, morpholine, or dioxane. In some embodiments, the heterocyclyl is a saturated or partially saturated heterocycle consisting of atoms selected from hydrogen, C, N, O, and S.

[0129] In some embodiments, aryl is C 6-24 In some embodiments, the aryl is C 6-16 In some embodiments, the aryl is C 6-10It is an aromatic ring. In some embodiments, aryl is phenyl, naphthyl, phenanthrenyl, anthracenyl, fluoroanthenyl, pyrenyl, chrysenyl, benzo[a]pyrenyl, pyrenyl, or coronenyl. In some embodiments, aryl is phenyl, naphthyl, or pyrenyl. In some embodiments, heteroaryl is a 3- to 24-membered aromatic heterocycle. In some embodiments, heteroaryl is a 3- to 15-membered aromatic heterocycle. In some embodiments, heteroaryl is a 3- to 10-membered aromatic heterocycle. In some embodiments, heteroaryl is a 3- to 6-membered aromatic heterocycle. In some embodiments, heteroaryl is an aromatic heterocycle consisting of atoms selected from hydrogen, C, N, O, and S.

[0130] In some embodiments, the substituted cycloalkyl, substituted heterocyclyl, substituted aryl, or substituted heteroaryl is selected from the group consisting of oxo, —COOH, —CONH 2 , —NH 2 , —NH 3 + , -NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH3, -SH, -S(O)CH3, -S(O)2CH3, and -S(O)2OH.

[0131] In some embodiments, the polymer has the structure of formula YA: [ka] or a pharmaceutically acceptable salt thereof; A 1 is hydrogen or methyl; A 2 is unsubstituted or substituted C 1-20 heteroalkyl; and a is an integer of 3 to 400 (for example, 3 to 200 (e.g., 40 to 120)).

[0132] In some embodiments, each A 2are identical (i.e., the polymer contains a single repeating monomer). In some embodiments, each A 2 are not identical (i.e., the polymer comprises multiple different monomers). 1 is hydrogen. In some embodiments, A 1 is methyl. In some embodiments, A 2 is an acid, ester, carboxamide, or substituted amide. In some embodiments, the polymer comprises 3 to 400 monomers (e.g., A1-A12) selected from: [ka] Here, a is an integer between 0 and 100.

[0133] In some embodiments, a is an integer between 3 and 400. In some embodiments, a is an integer between 10 and 200. In some embodiments, a is an integer between about 10 and about 30. In some embodiments, a is an integer between about 30 and about 60. In some embodiments, a is an integer between about 60 and about 90. In some embodiments, a is an integer between about 90 and about 120. In some embodiments, a is an integer between about 120 and about 150. In some embodiments, a is an integer between about 40 and about 120. In some embodiments, a is 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.

[0134] In some embodiments, the polymer has the structure of formula YB: [ka] or a pharmaceutically acceptable salt thereof; A 1 and B 1 is independently selected at each instance from hydrogen and methyl; A 2 and B 2 is, independently in each instance, C 1-20 heteroalkyl; and In each example, a and b are independently an integer of 0 to 400 (for example, 0 to 200 (for example, 40 to 120)), provided that the total number of monomer units is 3 or more.

[0135] In some embodiments, each A 2 are identical, and each B 2 are identical. 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), where each A 2 is the same in each example, and each B 2 is the same in each example, but each B 2 Each A 2 In some embodiments, each A 2 and B 2 is C 1-10 In some embodiments, each A is heteroalkyl. 2 is an amide, and each B 2 is an ester. In some embodiments, each A 2 is an ester, and each B 2 is an amide. In some embodiments, each A 2 and B 2 is the formula -C(O)OR 2 or -C(O)NR 4 R 5 C with 1-20 heteroalkyl group, where each R 2 , R 4 and R 5is as defined herein. In some embodiments, each A 2 and B 2 is the formula -C(O)OR 12 or -C(O)NR 14 R 15 C with 1-20 heteroalkyl group, where each R 12 , R 14 and R 15 is as defined herein.

[0136] In some embodiments, the polymer of formula YB comprises the following monomers: [ka]

[0137] In some embodiments, the polymer has the structure of formula Y-AB: [ka] or a pharmaceutically acceptable salt thereof; A 1 and B 1 is independently selected at each instance from hydrogen and methyl; A 2 and B 2 is, independently in each instance, C 1-20 heteroalkyl; In each example, a, b, and e are independently an integer of 0 to 400 (for example, 0 to 200 (for example, 40 to 120)), provided that the total number of monomer units is 3 or more.

[0138] In some embodiments, the polymer of formula Y-AB is a polymer comprising two adjacent monomers forming a repeat unit, where each A 2 is the same in each example, and each B 2 is the same in each example, but each B 2 Each A 2In 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, in each instance, are integers selected from 0 to 400 (e.g., a random copolymer). In some embodiments, each A 2 and B 2 is C 1-10 In some embodiments, each A is heteroalkyl. 2 is an amide, and each B 2 is an ester. In some embodiments, each A 2 is an ester, and each B 2 is an amide. In some embodiments, each A 2 and B 2 is the formula -C(O)OR 2 or -C(O)NR 4 R 5 C with 1-20 heteroalkyl group, where each R 2 , R 4 and R 5 is as defined herein. In some embodiments, each A 2 and B 2 is the formula -C(O)OR 12 or -C(O)NR 14 R 15 C with 1-20 heteroalkyl group, where each R 12 , R 14 and R 15 is as defined herein.

[0139] In some embodiments, the polymer has the structure of formula YC: [ka] or a pharmaceutically acceptable salt thereof; A 1 , B 1 and C 1 is independently selected at each instance from hydrogen and methyl; A 2 , B2 and C 2 is, independently in each instance, C 1-20 heteroalkyl; In each instance, a, b, c, and e are independently an integer selected from 0 to 400 (for example, 0 to 200 (for example, 40 to 120)), provided that the total number of monomer units is 3 or more.

[0140] In some embodiments, the polymer of formula YC is a polymer consisting of two adjacent monomers forming a repeat unit, where each A 2 is the same in each example, and each B 2 is the same in each example, but each B 2 Each A 2 and each C 2 is A 2 Same as B 2 and the monomer side chains, which can be the same as or different from both.

[0141] In some embodiments, each A 2 , B 2 and C 2 is C 1-10 In some embodiments, each A is heteroalkyl. 2 is an amide, and each B 2 is an ester, and each C 2 is an ester. In some embodiments, each A 2 is an amide, and each B 2 is an ester, and each C 2 is an amide. In some embodiments, each A 2 is an ester, and each B 2 is an amide, and each C 2 is an ester. In some embodiments, each A 2 is an ester, and each B 2 is an amide, and each C 2 is an amide. In some embodiments, each A 2 , B 2 and C 2 is the formula -C(O)OR 2 or -C(O)OR2 C with 1-20 heteroalkyl group, where each R 2 , R 4 and R 5 is as defined herein. In some embodiments, each A 2 , B 2 and C 2 is the formula -C(O)OR 12 or -C(O)NR 14 R 15 C with 1-20 heteroalkyl group, where each R 12 , R 14 and R 15 is as defined herein.

[0142] In some embodiments, the polymer of formula YC is: [ka]

[0143] Similar to the formula Y-AB, in some embodiments, the polymer has the structure of the formula Y-ABC: [ka] or a pharmaceutically acceptable salt thereof; A 1 , B 1 and C 1 is independently selected at each instance from hydrogen and methyl; A 2 , B 2 and C 2 is, independently in each instance, C 1-20 heteroalkyl; and In each instance, a, b, c, and f are independently an integer selected from 0 to 400 (for example, 0 to 200 (for example, 40 to 120)), provided that the total number of monomer units is 3 or more.

[0144] 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 trimeric repeat units (where 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 from 0 to 400 (e.g., 0 to 200, e.g., 40 to 120), provided that the total number of monomer units is 3 or greater. In some embodiments, each A 2 , B 2 and C 2 is C 1-10 In some embodiments, each A is heteroalkyl. 2 , B 2 and C 2 is an amide. In some embodiments, each A 2 , B 2 and C 2 is an ester. In some embodiments, the polymer has a ratio of amide to ester side chains of about 2:1.

[0145] In some embodiments, the polymer has the structure of formula YD: [ka] or a pharmaceutically acceptable salt thereof; A 1 , B 1 , C 1 and D 1 is independently selected at each instance from hydrogen and methyl; A 2 , B 2 , C 2 and D 2 is, independently in each instance, C 1-20 heteroalkyl; a, b, c, d, e and f are each independently an integer selected from 0 to 400 (for example, 0 to 200 (for example, 40 to 120)), provided that the total number of monomer units is 3 or more.

[0146] 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 trimeric repeat units and a second block having monomeric repeat units. In other embodiments, f is 1, and the polymer comprises 1, 2, 3, or 4 monomeric repeat units (side chain A 2 , B 2 , C 2 , D 2 In some embodiments, each A 2 , B 2 , C 2 and D 2 is an amide. In some embodiments, each A 2 , B 2 , C 2 and D 2 is an ester.

[0147] In some embodiments, the polymer of formula YD is: [ka]

[0148] 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 monomers in a given polymer chain, hi 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.

[0149] In some embodiments, the polymer comprises 3 or more monomer units, hi some embodiments, the polymer comprises about 5 or more monomer units, about 10 or more monomer units, about 15 or more monomer units, about 20 or more monomer units, about 30 or more monomer units, about 50 or more monomer units, about 100 or more monomer units, about 200 or more monomer units, about 300 or more monomer units, or about 400 or more monomer units.

[0150] In some embodiments, the polymer comprises from about 3 monomer units to about 400 monomer units, 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 400 monomer units, units to about 30 monomer units, about 4 monomer units to about 50 monomer units, 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 1 to about 120 monomer units, about 10 to about 150 monomer units, about 10 to about 200 monomer units, about 10 to about 400 monomer units, about 30 to about 50 monomer units, about 30 to about 80 monomer units, about 30 to about 100 monomer units, about 30 to about 120 monomer units, about 30 to about 150 monomer units, about 30 to about 200 monomer units, about 30 to about 400 monomer units, about 50 to about 80 monomer units 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,The polymer comprises about 100 to about 150 monomer units, about 100 to about 200 monomer units, about 100 to about 400 monomer units, about 120 to about 150 monomer units, about 120 to about 200 monomer units, about 120 to about 400 monomer units, about 150 to about 200 monomer units, about 150 to about 400 monomer units, or about 200 to about 400 monomer units. In some embodiments, the polymer comprises about 3 monomer units, 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. In some embodiments, the polymer comprises at least about 3, 4, 10, 30, 50, 80, 100, 120, 150, or 200 monomer units. In some embodiments, the polymer comprises at most about 4, 10, 30, 50, 80, 100, 120, 150, 200, or 400 monomer units. Each monomer unit is represented by C, 1-20 Contains heteroalkyl side chains.

[0151] In some embodiments, the polymer is a polyacrylate, also referred to as a poly(acrylic acid ester) or a poly(acrylic acid alkyl ester). In some embodiments, the polymer has monomer units comprising acrylates, also referred to as acrylic acid esters or acrylic acid alkyl esters. In some embodiments, the polymer is a polyacrylamide. In some embodiments, the polymer has monomer units comprising acrylamide (also referred to as acrylic amide). In some embodiments, each A 2 , B 2 , C 2 and D 2comprises an acrylate or an acrylamide.

[0152] In some embodiments, the monomer unit has a structure represented by the following formula: [ka] or a pharmaceutically acceptable salt thereof; R 1 and R 3 is independently selected at each instance from hydrogen and methyl; and Each R 2 is hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl, C 1-20 heteroalkyl, or a polyethylene glycol chain containing 1 to 100 ethylene glycol monomers; 1-6 Alkyl, C 7-20 Aralkyl, and C 1-20 Heteroalkyl is unsubstituted or substituted with one or more groups, wherein each of said 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 R 4 and R 5 each independently represents 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 heteroalkyl is unsubstituted or substituted with one or more groups, wherein each of said one or more groups is independently selected from the group consisting of 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.

[0153] In some embodiments, R 4 is hydrogen. In some embodiments, R 2 and R 5 is independently in each instance a substituted or unsubstituted C 1-6 In some embodiments, R 2 and R 5 are each independently a substitution C 1-6 alkyl, wherein the alkyl group is selected from -NH2, -NH3 + , -NHC(NH2 + )NH2, -NHCH3, -N(CH3)2, -N(CH3)3 + , -OH, -OCH, -S(O)CH, -S(O)CH, and -S(O)OH, or a pharmaceutically acceptable salt thereof. 2 and R 5 is independently selected from aminoalkyl, hydroxyalkyl, carboxyalkyl, alkoxy, haloalkyl, or any combination thereof. 2 is hydrogen. In some embodiments, R 4 is hydrogen. In some embodiments, R 5 is a substituted or unsubstituted C 1-6 an aminoalkyl group, where the aminoalkyl group is substituted, such as -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.

[0154] In some embodiments, the monomer unit has a structure represented by the following formula: [ka] or a pharmaceutically acceptable salt thereof; R 11 and R 13 is independently selected at each instance from hydrogen and methyl; and R 12 , R 14 and R 15 is, independently in each instance, 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 with one or more groups, and each group is 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 a pharmaceutically acceptable salt thereof; Or, R 14 and R 15 taken together form a substituted or unsubstituted heterocycle.

[0155] In some embodiments, R 14 is hydrogen. In some embodiments, R 14 and R 15 taken together form a substituted or unsubstituted heterocyclic ring (e.g., a 3- to 10-membered heterocycloalkyl ring). 14 and R 15taken together form 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 azocane, etc. In some embodiments, R 14 is hydrogen and R 15 is C 1-6 alkyl, where C 1-6 The alkyl is substituted with a heterocycle, such as 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 azocane, and the like.

[0156] In some embodiments, the polymer has a structure of formula YB: [ka] Here, each monomer unit (i.e., each of the following) [ka] has a structure represented by one of the following formulas: [ka] where each R 1 , R 2 , R 3 R 4 , R 5 , R 11 , R 12 , R 13 R 14 and R 15 is as defined above.

[0157] In some embodiments, the polymer has a structure of formula Y-B1 or formula Y-B2: [ka]

[0158] In some embodiments, the monomer units are selected from the group consisting of: [ka] Functional group (“Z”)

[0159] The compounds disclosed herein include a functional group "Z" attached to a polymer "Y", linked 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, functional groups disclosed herein generally include sulfur groups (e.g., -SR 6 In some embodiments, the functional group comprises a reactive group, a charged group, a detectable group, a capping group, or a combination thereof.

[0160] The functional group may be or may 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.

[0161] In some embodiments, the functional group is a thiol or sulfhydryl, e.g., -SH. In some embodiments, the functional group is a sulfide, e.g., -SR. 6 In some embodiments, the functional group is a hydroxyl, e.g., -OH. In some embodiments, the functional group is an ether, e.g., -OR. 6 In some embodiments, the functional group is a sulfoxide, e.g., —S(O)R 6 , or sulfones, e.g., S(O)R 6 is.

[0162] 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, R6 is a heteroalkyl group. In some embodiments, R 6 is C 1-100 In some embodiments, R 6 is C 1-20 In some embodiments, R 6 contains a sulfur atom linked by alkyl or heteroalkyl groups, each of which has from 1 to about 200 atoms, where 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, etc., are considered within the scope of the present invention.

[0163] In some embodiments, R 6 comprises a reactive group, a charged group, a detectable group, a peptide group, a capping group, or a combination thereof.

[0164] In some embodiments, R 6 The reactive group in R comprises an azide or an alkyne. 6 can react with another molecule containing an alkyne or azide via "click" chemistry or "click" reactions, particularly [3+2] cycloaddition reactions, 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), etc. In some embodiments, the alkyne is a cyclooctyne, such as dibenzylcyclooctyne, biarylazacyclooctynone, and fluorinated cyclooctyne, or a bicyclononyne.

[0165] In some embodiments, R 6 In some embodiments, the charged groups in R include one or more cationic groups. 6 In some embodiments, the one or more cationic groups in R include a cyclic amine, a primary amine, a guanidine, or a combination thereof. 6The charged group may include multiple cationic groups (e.g., three or more cationic groups). The cationic groups described herein may include amines or other organic groups capable of stably maintaining a positive or partially positive charge at physiologically relevant pH. For example, primary amines, secondary amines, tertiary amines, or quaternary amines are all considered cationic groups. In particular, quaternary amines have a positive (cationic) charge regardless of their protonation state and may be useful in preparing the compounds described herein.

[0166] In some embodiments, R 6 The detectable group of R comprises a fluorophore, dye, or Förster resonance energy transfer (FRET) donor or acceptor. In some embodiments, a fluorophore (also called a fluorescent dye, chromophore, or fluorescent probe), dye, or FRET donor or acceptor is a fluorescent compound that can re-emit light upon light 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 In some embodiments, the detectable group in R comprises a fluorescein compound. 6 In some embodiments, the detectable group in R comprises a rhodamine compound. 6 The detectable group comprises a coumarin compound. In some embodiments, the detectable group comprises a derivative of a fluorescein, rhodamine, or coumarin compound. In some embodiments, the detectable group comprises or is derived from a fluorescent azide (e.g., azidefluor-488).

[0167] In some embodiments, the detectable group comprises a near-infrared fluorophore, 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 contemplated within the scope of the present invention include molecules or portions of molecules that emit a signal in response to an excitation source including infrared, near-infrared, visible, or ultraviolet light.

[0168] In some embodiments, R 6 The capping group is an inert group. In some embodiments, the inert group is a chemically inert group or a chemically unreactive group. In some embodiments, the capping group is a hydrocarbon (e.g., a C group such as a methyl group, an ethyl group, or a propyl group). 1-20 In some embodiments, the capping group is a thiol.

[0169] The functional group may include a binding group. For example, the binding group may be a small molecule capable of binding other atoms or molecules. The binding group may include a chelating agent configured to bind atoms such as metals. In a specific example, the compound may 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 may be an ion or an oxide. Specifically, the metal atom is a lanthanide or 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.

[0170] The functional group may also include a peptide group. In some cases, the functional group includes a targeting group (e.g., when the targeting group is a peptide group). The targeting group may be a peptide. The targeting group may be a cyclic peptide. In a specific example, the functional group targets an integrin receptor (e.g., α vThe functional group may include cyclic peptides that target the β3-integrin receptor. For example, the functional group may include 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 a specific example, a functional group containing a cyclo(-Arg-Gly-Asp-D-Phe-Lys) ("cRGDfK") group was synthesized. Radiolabeled analogs, salts, or radioisotope derivatives thereof are also included within the scope of the present invention. In examples having a functional group containing a cRGDfK group, the cRGDfK group may be linked to the polymer (Y), for example, via a lysine nitrogen atom, optionally via a linker. The term "linker," as used herein, includes any alkyl group, heteroalkyl group, cyclyl group, heterocyclyl group, or any combination thereof, containing 1 to 100 atoms selected from C, H, N, O, and S. The linker may include, for example, a maleimide, an ethylene unit, a propylene unit, an amide, an amine, an ester, an ether, a thioether, a polyethylene glycol chain, an alkyl chain, a click reagent, a peptide, or any combination thereof. In some embodiments, the functional group (optionally including the linker) is any one of the groups (Z) shown in Table 1.

[0171] The functional group may comprise a therapeutic group. For example, the functional group may comprise a drug linked via a cleavable linker. The therapeutic group may also be a binding agent or a radiotherapeutic group comprising a chelator and a radioisotope, as described above. In some embodiments, the functional group comprises a chelating group. The chelating group may coordinate with a metal ion. The chelating group is useful as a therapeutic agent or a detectable agent (in PET or SPECT imaging). The chelating group may be cyclic or acyclic and often comprises two or more basic amines or acidic carboxylates. One example of a chelating functional group is the aminopolycarboxylic acid group, nitrilotriacetic acid (NTA). Additional aminopolycarboxylic acid chelators include NTA, EDTA, DTPA, EGTA, BAPTA, NOTA, DOTA, and their derivatives.

[0172] In some embodiments, the functional group (Z) is selected from:

[0173] [ka] Here, the functional group is attached to the polymer via the sulfur atom on the left side of each of the above structures.

[0174] In another aspect, provided herein is a compound of Formula I: XYZ or a pharmaceutically acceptable salt thereof; X is a lipid selected from XA, XB, and XC: [ka] Y is a polymer having a structure according to formula YA, YB, YC, or YD; and Z is a functional group shown below.

[0175] Certain embodiments of the present invention are disclosed in Table 1 below. Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0176] In some embodiments, the compounds are configured to encapsulate or complex nucleic acids in aqueous solution. In some embodiments, the compounds are substantially non-toxic.

[0177] 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 a molecular weight of 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 10 kDa to about In some embodiments, the compound comprises a molecular weight of about 100 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 70 kDa, about 20 kDa to 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, or about 90 kDa, hi 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. Liposomes and Nanoparticles

[0178] The lipid-polymer compounds disclosed herein can be useful in preparing liposomes and / or lipid nanoparticles. For example, the PLips disclosed herein can serve a variety of purposes. For example, the PLips disclosed herein can add stability or other benefits to lipid nanoparticles (LNPs). Lipid nanoparticles are generally spherical vesicles made from ionizable lipids that are positively charged at low pH (enabling complexation with RNA) 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 ionizability of lipids at low pH facilitates endosomal escape, allowing the release of the payload into the cytoplasm of the target cell. The PLips disclosed herein can be used to prepare lipid nanoparticles (LNPs). Thus, the use of the PLips disclosed herein in preparing lipid nanoparticles, as well as any lipid nanoparticles containing the PLips (or "compounds") disclosed herein, is within the scope of the present invention. The PLips disclosed herein can aid in the stability of LNPs (e.g., stabilized PLips). In many cases, PLips can have multiple utilities. For example, a PLip can be both a stabilized PLip and a reactive PLip, meaning that the PLip has a stabilized polymer block and the functional group contains a reactive moiety (e.g., a strained cyclooctyne or azide). Similarly, a PLip can be cationic and contain a reactive moiety. In some cases, the reactive moiety reacts with a dye to form a fluorescently labeled LNP. Stabilized PLips can be used as an alternative to PEG, which has demonstrated toxicity and problematic side effects in some biological systems. Stabilized PLips may contain PEG side chains or heteroalkyl acrylate or acrylamide side chains.

[0179] Cationic PLips can contain multiple cationic groups. In some cases, cationic PLips contain about 3 to about 20 cationic monomer units. In some embodiments, cationic PLips can replace cationic lipids used in LNP formulations. Cationic PLips can be useful as transfection reagents (e.g., the cationic amine (N) interacts noncovalently with the phosphate backbone (P) of nucleic acids). 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 certain examples, the N:P ratio is about 1:4 to about 128:1. In more specific examples, the N:P ratio is about 4:1 to about 16:1. The cationic PLip can have a positive charge. In some examples, the cationic PLip has 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 positive charges. The positive charge can be directly correlated with the polymer length of the polymer containing the cationic side chains. For example, a cationic PLip having a polymer containing 19 cationic monomer units can have a charge of +19. Similarly, a cationic PLip having a polymer containing 13 cationic monomer units can have a charge of +13. In other examples, the polymer can include two blocks of repeating monomer units, where the first block includes the stabilizing side chains and the second block includes the cationic side chains. In such cases, the stabilizing polymer block may exceed the length of the cationic block by 2x, 3x, 4x, 5x, 8x, 10x, 16x, 20x, 32x, 50x, 64x, or 100x or more. For example, in some cases, the polymer comprises a stabilizing block of about 100 to about 200 (e.g., about 170 to about 190) monomer units and a second cationic monomer unit block comprising about 3 to about 30 cationic monomer units (e.g., about 3 to about 10 monomer units).In another embodiment, the polymer comprises alternating blocks of monomer units, where a first monomer unit has a stabilizing (uncharged) side chain and a second monomer unit has a cationic side chain.

[0180] The lipid-polymer compounds (e.g., cationic PLips) disclosed herein may be useful in preparing nanoparticles and / or transfection reagents. As used herein, "nanoparticle" generally refers to an aggregate of compounds capable of forming vesicles. As described herein, nanoparticles can be configured to encapsulate one or more nucleic acid molecules, thereby forming transfection reagents, i.e., reagents configured to deliver genetic information to host cells. Nanoparticles can be configured, for example, to encapsulate or complex with nucleic acids (e.g., DNA, RNA, etc.). Nanoparticles prepared as described herein can be configured to encapsulate or complex with the nucleic acid at a ratio of 0.3:1 to 100:1 (weight:weight). For example, transfection reagents may encapsulate or complex one or more nucleic acids at a weight:weight ratio of 0.5:1 to 100:1, 1:1 to 100:1, 5:1 to 100:1, 10:1 to 100:1, 20:1 to 100:1, 30:1 to 100:1, 40:1 to 100:1, 50:1 to 100:1, 60:1 to 100:1, 70:1 to 100:1, 80:1 to 100:1, 90:1 to 100:1, or 95:1 to 100:1. Generally, transfection reagents refer to the combination of nanoparticles and genetic material, while nanoparticles refer to the vesicles themselves. Furthermore, nanoparticles may contain multiple compounds or PLips disclosed herein. The advantages of nanoparticles and transfection reagents containing compounds or PLips disclosed herein are apparent from the examples.

[0181] In some embodiments, encapsulation or complexation of a nucleic acid increases the half-life of the nucleic acid under aqueous or physiological conditions by at least 2. In some embodiments, encapsulation or complexation of a nucleic acid increases the half-life of the nucleic acid 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.

[0182] 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 transfection complexes with an average size of 20 to 2,000 nm. In some embodiments, the encapsulation or complexation of the nucleic acid produces transfection complexes with an average size of about 20 nm to about 2,000 nm.In some embodiments, the encapsulation or complexation of nucleic acids is carried out in a manner that the average size is 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 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 Approx. 300nm, approx. 50nm ~ approx. 400nm, approx. 50nm ~ approx. 500nm, approx. 50nm ~ approx. 1,000nm, approx. 50nm ~ approx. 1,500nm, approx. 100nm to about 400nm, about 100nm to about 500nm, about 100nm to about 1,000nm, about 100nm to about 1,500nm, about 100nm to about 2,000nm, about 200nm to about 300nm, about 200nm to about 400nm, about 20 0nm to about 500nm, about 200nm to about 1,000nm, about 200nm to about 1,500nm, about 200nm to about 2,000nm, about 300nm to about 400nm, about 300nm to about 500nm, about 300nm to about 1,000nm, about 300nm nm to about 1,500 nm, about 300 nm to about 2,000 nm, about 400 nm to about 500 nm, about 400 nm to about 1,000 nm, about 400 nm to about 1,500 nm, about 400 nm to about 2,000 nm, about 500 nm to about 1,000 nm, about 500 nm to about 1,500 nm, about 500 nm to about 2,000 nm, about 1,000 nm to about 1,500 nm, about 1,000 nm to about 2,000 nm, or about 1,500 nm to about 2,000 nm. In some embodiments, encapsulation or complexation of the nucleic acid produces transfection complexes with 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, encapsulation or complexation of nucleic acids produces transfection complexes with 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, encapsulation or complexation of nucleic acids produces transfection complexes with 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, encapsulation or complexation of nucleic acids produces transfection complexes with an average radius of about 100 nm to about 300 nm (e.g., about 100 nm 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, the temperature-sensitive LNPs can induce temperature-dependent swelling of the LNPs, e.g., from a radius of about 180 nm to a radius of about 300 nm. Swelling of the temperature-sensitive LNPs can, in some instances, be a reversible process (e.g., whereby 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 disintegrate or aggregate after heating (e.g., to about 45°C). This process can be completely or nearly completely reversible (i.e., less than about 2% loss of LNPs per thermal cycle).

[0183] In some embodiments, encapsulation or complexation comprises adsorption of at least a subset of the nucleic acids to a surface of the transfection reagent. In some embodiments, encapsulation or complexation of nucleic acids produces transfection complexes configured for cellular uptake. In some embodiments, cellular uptake comprises endocytosis.

[0184] In some embodiments, encapsulation or complexation of the nucleic acid produces a transfection complex with an aqueous solubility of at least 5 μg / mL. In some embodiments, encapsulation or complexation of the nucleic acid produces a transfection complex with an aqueous 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.

[0185] In some embodiments, encapsulation or complexation of nucleic acid produces a transfection complex with an aqueous solubility of about 10 μg / mL to about 50 μg / mL, hi some embodiments, encapsulation or complexation of nucleic acid produces a transfection complex with an aqueous solubility of about 1 μg / mL to about 100 μg / mL. In some embodiments, encapsulation or complexation of nucleic acids is from about 1 μg / mL to about 5 μg / mL, from about 1 μg / mL to about 10 μg / mL, from about 1 μg / mL to about 20 μg / mL, from about 1 μg / mL to about 30 μg / mL, from about 1 μg / mL to about 40 μg / mL, from about 1 μg / mL to about 50 μg / mL, from about 1 μg / mL to about 100 μg / mL, from about 5 μg / mL to about 10 μg / mL, from about 5 μg / mL to about 20 μg / mL, from about 5 μg / mL to about 30 μg / mL, from about 5 μg / mL to about 40 μg / mL, from about 5 μg / mL to about 50 μg / mL, from about 5 μg / mL to about 100 μg / mL, from about 10 μg / mL to about 20 μg / mL, from about 10 μg / mL to about 3 Generate transfection complexes with water solubility of 0 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, encapsulation or complexation of the nucleic acid produces a transfection complex with an aqueous 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, encapsulation or complexation of the nucleic acid produces a transfection complex with an aqueous 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, encapsulation or complexation of the nucleic acid produces a transfection complex with an aqueous 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.

[0186] In some embodiments, encapsulation or complexation of nucleic acid produces a transfection complex with an aqueous solubility of about 5 μg / mL to about 5,000 μg / mL. In some embodiments, encapsulation or complexation of nucleic acid produces a transfection complex with an aqueous 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 50 μg / mL to about 500μg / mL, approximately 50μg / mL to approximately 1,000μg / mL, approximately 50μg / mL to approximately 2,000μg / mL, approximately 50μg / mL to approximately 3,000μg / mL, approximately 50μg / mL to approximately 4,000μg / mL, approximately 50μg / mL to approximately 5 ,000μg / mL, about 100μg / mL to about 500μg / mL, about 100μg / mL to about 1,000μg / mL, about 100μg / mL to about 2,000μg / mL, about 100μg / mL to about 3,000μg / mL, about 100μg / m L ~ approx. 4,000 μg / mL, approx. 100 μg / mL ~ approx. 5,000 μg / mL, approx. 500 μg / mL ~ approx. 1,000 μg / mL, approx. 500 μg / mL ~ approx. 2,000 μg / mL, approx. 500 μg / mL ~ approx. 3,000 μg / mL, approx. 00μg / mL ~ approx. 4,000μg / mL, approx. 500μg / mL ~ approx. 5,000μg / mL, approx. 1,000μg / mL ~ approx. 2,000μg / mL, approx. 1,000μg / mL ~ approx. 3,000μg / mL, approx. 1,000μg / mL ~ approx. 4 Generate transfection complexes with aqueous solubilities of about 1,000 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 the nucleic acid produces a transfection complex with an aqueous 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 the nucleic acid produces a transfection complex with an aqueous 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 the nucleic acid produces a transfection complex with an aqueous 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.

[0187] In some embodiments, the compound has a dispersity (i.e., molecular weight distribution) of about 2.0 or less. In some embodiments, the compound has a dispersity of about 1.5 or less. In some embodiments, the compound has a dispersity of about 1.3 or less. In some embodiments, the compound has a dispersity of about 1.2 or less. In some embodiments, the compound has a dispersity of at least 1.0. In some embodiments, the compound has a dispersity of at least 1.1. In some embodiments, the compound has a dispersity of at least 1.2. In some embodiments, the compound has a dispersity of at least 1.3. In some embodiments, the compound has a dispersity of at least 1.4. In some embodiments, the compound has a dispersity of at least 1.5. In some embodiments, the compound has a dispersity of about 1.1 to about 1.5. In some embodiments, the compound has a dispersity of about 1.2 to about 1.5. In some embodiments, the compound has a dispersity of about 1.3 to about 1.5. In some embodiments, the compound has a dispersity of about 1.2 to about 1.8. In some embodiments, the compound has a dispersity of about 1.5 to about 1.8. In some embodiments, the compound has a dispersity of about 1.5 to about 2.0. In some embodiments, the compound has a dispersity of about 1.8 to about 2.0. In some embodiments, the compound has a dispersity of about 1.8 to about 2.3. In some embodiments, the compound has a dispersity of about 2.0 to about 2.3. In some embodiments, the compound has a dispersity of about 2.0 to about 2.5. In some embodiments, the compound has a dispersity 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. Transfection reagents

[0188] In one aspect, provided herein is a reagent (e.g., a transfection reagent) comprising a compound disclosed herein and a nucleic acid. The transfection reagent can act as a delivery vehicle for a payload (e.g., a biologically active compound (e.g., a nucleic acid)). The payload may be specifically combined with a particular transfection reagent, or the transfection reagent can deliver various payloads. The transfection reagent can provide multiple advantageous properties to aid in the efficient delivery of the payload to a target (e.g., a target cell). For example, the transfection reagent can include a liposome or lipid nanoparticle (LNP). Liposomes and LNPs can act as microvesicles that are roughly spherical and contain an interior and an exterior. Advantageously, encapsulating the payload within the liposome or LNP can protect it from biological and chemical processes that may degrade the payload. Alternatively, the encapsulated payload can reside within a lipid bilayer membrane rather than within an internal compartment. In yet another example, the internal compartment can be tailored to stabilize a specific payload.

[0189] In another example, the transfection reagent forms a lipid bilayer around the payload, and upon contact with the target cell, it fuses with the cell and releases the payload into the cell. The transfection reagent can also physically exclude an enzyme or ribozyme, or shield the internal payload from external environmental changes (e.g., pH, ion concentration, etc.).

[0190] In another aspect, transfection reagents containing the compounds disclosed herein can directly interact with the payload, for example, stabilizing or chelating specific groups. For example, transfection reagents containing the compounds disclosed herein can form polar, ionic, or other non-covalent interactions with the nucleic acid payload. While some vesicles may contain a simple lipid shell and an aqueous internal compartment, other vesicles (e.g., LNPs) can distribute compounds within the internal space (e.g., interact with the internalized payload). In certain examples, transfection reagents containing the compounds disclosed herein (a) generate vesicles that separate the internal compartment from the external environment and (b) encapsulate the internal payload with the compounds disclosed herein. Particularly useful are polymers disclosed herein that can include a hydrocarbon backbone capable of forming a bilayer, while the polymer side chains can form stabilizing interactions with the nucleic acid payload (e.g., the phosphate backbone of the nucleic acid payload). The nucleic acid payload can include DNA or RNA (e.g., siRNA, saRNA, mRNA, microRNA, etc.).

[0191] In yet another aspect, the transfection reagents disclosed herein can include lipid-polymer compounds having functional groups that act as reporter or reactive moieties. In addition to the aforementioned aspects of stabilizing, shielding, and delivering a payload into the intracellular space of a target cell, transfection reagents containing reporter or reactive compounds can fuse with target cells, thereby being internalized or inserted into the target cells. In a specific example, a transfection reagent containing a polymer-lipid compound having a fluorescent functional group can deliver a nucleic acid payload to a target cell and, upon fusing with the target cell, label the target cell with the fluorescent functional group. Similarly, a transfection reagent containing a reactive functional group (e.g., a click handle) can contact and insert into a target cell, thereby providing a reactive handle to the target cell. Additional functional groups disclosed herein can similarly be incorporated into target cells, thereby functionalizing the target cell with the functional group.

[0192] The nucleic acid can 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 can be 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 2k b ~ about 100kb, about 5kb - about 8kb, about 5kb - about 10kb, about 5kb - about 12kb, about 5kb - about 15kb, about 5kb - about 20kb, about 5kb - 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 50 kb, 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 10kb to about The nucleic acid may comprise about 1 kb, about 2 kb, about 5 kb, about 8 kb, about 10 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. 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 between about 2 kb and about 20 kb (e.g., between about 5 kb and about 15 kb (e.g., between about 8 kb and about 12 kb)). In a specific example, the nucleic acid comprises about 10 kb.

[0193] Chemical transfection of nucleic acids may offer a simple and robust alternative to viral, liposomal, and electroporation delivery. Upon complexation with nucleic acids, the physical properties of the resulting nucleic acid complexes may change over time, potentially affecting the functional performance of the complexes. In some cases, transfection complexes with a radius of 200–400 nm may be optimal for many applications. Complex formation time may be one of the key parameters that can be modified to control the size of transfection complexes for achieving nucleic acid delivery. While the functional performance of transfection complexes does not solely depend on the size of the complexes, dynamic light scattering (DLS) can be used to track the size changes of growing aggregates over time. Various factors, including the composition of the transfection reagent, salt, and pH, can affect the size and functional performance of the resulting complexes after nucleic acid addition.

[0194] In some cases, commercially available transfection reagent protocols specify an optimal complex formation time. For example, the recommended formation time for TransIT®-mRNA transfection complexes is less than 5 minutes, while that for TransIT-X2® is 15-30 minutes. However, in some cases, such as large-scale automated transfections, this time frame may be undesirable or impractical due to the time required for adding and mixing the transfection complexes. As a result, it is desirable to design a method for using stabilized polymer-lipid hybrids (PLips) in the transfection reagent formulation prior to complex formation with DNA as a way to control or extend the transfection complex formation time.

[0195] In some embodiments, the optimal complex formation concentration may be a factor of commercially available transfection reagent protocols. For example, transfection reagent complexes can be formed at an initial concentration 10 times higher than the concentration of the complex after addition to cells in the medium (assuming the complex is added at 10% vol / vol compared to the total volume of medium in which the cells are transfected). For a 200 L bioreactor, a typical transfection reagent would add 20 L of transfection complex. Adding this amount in a timely manner to maintain the functionality of the transfection reagent complex is difficult, so adding a smaller amount of complex is preferable (to reduce addition time and handling). Therefore, adding a higher concentration of complex is beneficial, but at higher concentrations, complex growth becomes more rapid and uneven, leading to quality issues (especially in a GMP environment). The addition of stabilized PLips may mitigate the growth of transfection complexes at higher concentrations. Stimulus-sensitive transfection

[0196] In some aspects, the lipid-polymer compounds provided herein contain a stimulus-sensitive moiety in the linker region between the lipid tail and the polymer head group containing the stabilizing monomer unit. These types of stimulus-sensitive stabilized PLips not only enhance the stability of the transfection reagent complex, but also control the timing of transfection reagent release within the reactor.

[0197] Transfection complexes are formed when nucleic acids are combined with transfection reagents (which can include a mixture of polymers and lipids). Through a combination of electrostatic and other noncovalent interactions, cationic, nonviral, nonliposomal formulations can bind to negatively charged nucleic acids, stabilizing and condensing them. For in vitro delivery, the net charge of the complex can enhance binding to the cell surface. A net positively charged complex is achieved when the number of moles of positively charged amines (N) available on the polymer head and / or lipid tail exceeds the number of moles of negatively charged phosphates (P) available on the nucleic acid. The N:P ratio is often calculated to determine whether the complex will have a net positive, neutral, or negative charge. When incubated with cells, the positively charged complex electrostatically interacts with the negatively charged cell membrane, allowing cellular uptake by endocytosis. Once endocytosed, the transfection complex may become trapped in endosomes, potentially leading to undesirable outcomes such as lysosomal degradation or transport out of the cell. The PLips disclosed herein are not only capable of stabilizing transfection reagents, but are also stimuli-responsive and can impart a higher positive charge to the complex when added to cells maintained at 37°C.

[0198] 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: Chemical composition of the transfection reagent Complex formation time between transfection reagent and nucleic acid The type and size of the nucleic acid to be delivered The medium in which the transfection complex is formed The molar charge ratio of the transfection reagent to the nucleic acid (often reported as the amine / phosphate charge ratio, N / P) Reagent and nucleic acid concentrations

[0199] Disclosed herein is a method for extending the optimal window for adding transfection complexes to cells from minutes to hours (potentially days) without altering the functional performance of the reagent. Electrostatic interactions between the transfection complex and cellular components are slowed, resulting in much slower growth of the transfection complex, or "stabilized" over time. Incorporation of stabilized PLips into transfection complexes (which most often contain nucleic acids, cationic polymers, and cationic lipids) occurs through hydrophobic interactions of their lipid tails. Once incorporated into the complex, the polymer headgroup of the PLip provides the "stabilizing" moiety. This polymer headgroup is a sufficiently long hydrophilic chain that stabilizes the complex by disrupting the electrostatic interactions that would otherwise cause the complex to continue growing over time.

[0200] Scheme 1 shows examples of the design of stabilized PLips and stimuli-responsive stabilized PLips. The difference between these two designs is that in stimuli-responsive stabilized PLips, a stimuli-responsive linker unit is inserted between the lipid tail and the stabilizing polymer head group. Other designs are possible. For example, multiple stimuli-responsive linker units can be incorporated into the PLip structure. For example, one of the monomer units of the stabilizing polymer head group may contain one or more stimuli-responsive linker units. Upon the 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, a chemical, pH, or other factors, and can cause a change in the properties of the PLip. The change can be from an extended or coiled form to a condensed form or globular aggregate; from a positive charge to a neutral or negative charge; from a neutral to a negative charge; from a neutral to a positive charge; from a negative charge to a neutral or positive charge; from a stable to an unstable form; etc.

[0201] Scheme 1:

number

[0202] Temperature responsive PLip

[0203] Particularly for in vitro transfection complexes, the particle's zeta potential and overall positive charge can affect delivery to negatively charged cell membrane surfaces. Therefore, the properties and concentration of stabilizing PLips may play a role. For example, adding too much stabilizing PLip may inhibit electrostatic interactions with cells as well as with other transfection complexes. Adding too little PLip may not achieve the desired effect of stabilizing complexes and extending the optimal time allowed for optimal size and transfection efficiency. Similarly, PLips with headgroups that are too long or too short may have similar effects.

[0204] In some embodiments, PLips can be stimuli-responsive. In some embodiments, temperature-sensitive PLips can contain: (i) a lipid tail end group at one end (to interact with transfection complexes), (ii) a temperature-sensitive linker unit group in the middle that includes a polymer moiety (e.g., poly(N-isopropylacrylamide) ("P(NIPAm)"), and (ii) a stabilizing polymer head group at the other end that includes another polymer moiety (e.g., PEG, poly(2-hydroxyethyl acrylate) ("PHEA"), or poly[oligo(ethylene glycol) methyl ether methacrylate] ("POEGMA")). Temperature-sensitive PLips can be used to take advantage of the fact that transfection complexes form at room temperature (25°C) or below, while cell transfection is performed at 37°C. Some polymers, such as P(NIPAm), may have a lower critical solution temperature (LCST) in water below 32°C, meaning that they are soluble in water (hydrophilic-like) at temperatures below this temperature, but transition to an insoluble (hydrophobic-like) state above this temperature. Above the LCST, P(NIPAm) polymer chains may not adopt an extended random coil conformation because the protons in the P(NIPAm) side chains do not form hydrogen bonds with surrounding water molecules but instead interact with protons in other P(NIPAm) side chains. This transition may cause P(NIPAm) to adopt a collapsed conformation, potentially excluding water molecules. When incorporated into transfection complexes, the net effect may be a more stable, less cationic complex at room temperature and a less stable, more cationic complex at 37°C. Scheme 2 below shows a schematic representation of the stabilized hydrodynamic volume associated with transfection complexes at 25°C and 37°C.

[0205] Scheme 2:

number

[0206] At 25 °C, the hydrodynamic volume is increased compared to 37 °C, potentially resulting in increased stability of the transfection complex. These properties may be a result of the extended P(NIPAm) linker unit at 25 °C and the presence of POEGMA units in the polymer head group. At 37 °C, the P(NIPAm) block collapses and is no longer hydrophilic, leaving only the outer POEGMA block as a stabilizing moiety.

[0207] In some embodiments, the temperature-responsive unit has a lower critical solution temperature (LCST) of about 27° C. to about 35° C. In some embodiments, the temperature-responsive unit has a lower critical 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(Nn-propylacrylamide), poly(N-methyl-Nn-propylacrylamide), poly(N,N-diethylacrylamide), poly(N-isobutylacrylamide), poly(N-sec-butylacrylamide), poly(Nn-butylacrylamide), poly(N-isobutylacrylamide), hydroxypropyl cellulose, poly(N-vinylcaprolactam), poly-2-isopropyl-2-oxazoline, or polyvinyl methyl ether, or a combination thereof. In some embodiments, the temperature-responsive unit comprises between 2 and 250 monomer units.

[0208] Another example of a stimuli-responsive polymer is pH-responsive Plip. In some embodiments, pH-responsive Plip stabilizes transfection complexes at pH 7.4, but may be less stable at lower pHs, such as those encountered in endosomes or other cells. pH-responsive Plips may swell, collapse, or undergo structural or morphological changes depending on the pH of their environment. This pH-dependent behavior may be due to the presence of specific pH-sensitive functional groups on the polymer chain. Sensitivity may be either acidic or basic, and they may respond to either basic or acidic pH conditions as stimuli. For example, polymers with acidic groups (such as -COOH and -SO3H) and polymers with basic groups (-NH2) may be pH-sensitive polymers. The response mechanism may be similar in both groups; only the stimuli differ.

[0209] Examples of polyacid polymers (anionic polymers) include acidic functional groups such as carboxylic acid (-COOH), sulfonic acid (-SO3H), phosphonic acid, and boronic acid. Therefore, polyacids can accept protons even at low pH values. At high pH values, they can deprotonate and become negatively charged. The negative charge creates a repulsive force, which can cause the polymer to swell. This swelling behavior is observed when the pH is higher than the pKa of the polymer. Examples of polyacid polymers include polymethyl methacrylate polymer and cellulose acetate phthalate. In some cases, polymethacrylic acid (PMAAc) can accept protons at low pH and release protons at neutral and high pH. Additional examples of polyacid polymers include poly(carboxylic acids), poly(phosphoric acids), poly(sulfonic acids), poly(amino acids), and poly(boronic acids).

[0210] Examples of polybase polymers include the basic equivalents of polyacid polymers, also known as cationic polymers. Like polyacid polymers, they can accept protons at low pH, but may subsequently become positively charged. In contrast, at high pH values, they are neutral. Swelling behavior may be observed when the pH is lower than the pKa of the polymer. Poly[(2-dimethylamino)ethyl methacrylate] (PDMA) can accept protons at low pH, potentially forming positively charged polymer chains. Other examples include polymers containing tertiary amine, morpholino, pyrrolidine, piperazine, pyridine, or imidazole groups as part of the polymer's side chains.

[0211] Other pH-sensitive polymers include alginate, chitosan, carboxymethylcellulose, carboxymethyldextran, gelatin A and B, and hyaluronic acid. How to use

[0212] Disclosed herein are methods for transfecting cells, the methods 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 contacting occurs under conditions suitable for 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 comprise conditions sufficient for iontotropic gelation.

[0213] In some embodiments, provided herein are methods of transfecting a cell, wherein the transfection complex comprises a positive charge under conditions suitable for entry of the nucleic acid into the cell.

[0214] In some embodiments, provided herein are methods of transfecting a cell, wherein the nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or any combination thereof.

[0215] The present invention provides a method for transfecting cells, wherein the contacting is for less than about 24 hours. In some embodiments, the contacting is 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.

[0216] In some embodiments, provided herein are methods for lipid-mediated transfection of a cell, comprising contacting the cell with a transfection reagent disclosed herein (e.g., a composition comprising a compound and a nucleic acid described herein). In some embodiments, provided herein are methods for preparing a compound (e.g., a lipid-polymer conjugate) or a transfection reagent comprising the same, as described in the Examples below. Abbreviation

[0217] Unless otherwise noted, abbreviations used throughout this disclosure are defined as follows: 1 H NMR proton nuclear magnetic resonance AIBN 2,2'-azobis(2-methylpropionitrile) AF488 azidefluor-488 C Celsius CPCPA 4-cyano-4-((phenylcarbonothioyl)thio)pentanoic acid DBCO Dibenzocyclooctyne DCM dichloromethane DIC Diisopropylcarbodiimide DLIN DLIN (6Z,9Z,28Z,31Z)-heptatriacon-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoic acid or DLin-MC3-DMA DLS Dynamic Light Scattering DMAP 4-dimethylaminopyridine DMF N,N-dimethylformamide DNA deoxyribonucleic acid DOPE Dioleoylphosphatidylethanolamine EtOH ethanol HEA Hydroxyethyl Acrylate HPLC High Pressure Liquid Chromatography HPMA N-(2-hydroxypropyl)methacrylamide LC / MS Liquid Chromatography / Mass Spectrometry LNP lipid nanoparticles MeOH Methanol MFI Mean Fluorescence Intensity MSEMA 2-(methylsulfinyl)ethyl methacrylate NI normalized intensity NIPAm N-isopropylacrylamide PEG-DMG 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol P(HPMA) Poly(N-(2-hydroxypropyl)methacrylamide) PLIP polymer-lipid compound RAFT Reversible Addition-Fragmentation Chain Transfer Polymerization RFU Relative Fluorescence Units SMFI specific mean fluorescence intensity TLC thin layer chromatography v / v capacity ratio [Example]

[0218] The following illustrative examples are representative of aspects of the compounds, compositions, and methods described herein, and are not intended to be limiting in any way. Synthesis Example

[0219] The compounds disclosed herein can be synthesized according to, or in analogy with, the following synthetic examples. Additionally, the examples disclosed herein can be adapted and modified according to principles known in the art to produce the various polymer-lipid compounds, synthetic intermediates (e.g., RAFT agents), and transfection reagents (e.g., LNPs) disclosed herein. Example S1: Synthesis and characterization of dioleyl RAFT agents [ka]

[0220] i. To a sample of 4-cyano-4-((phenylcarbonothioyl)thio)pentanoic acid ("CPCPA") (4.00 g, 14.3 mmol) in DMF (20 mL), excess glycerol (16.0 g, 0.118 mol) and diisopropylcarbodiimide (DIC, 2.80 mL, 17.9 mmol) were added and stirred at room temperature for 30 minutes. 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 layer. 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 it dropwise twice to water (80 mL). 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 on a rotary evaporator and redissolved in chloroform (10 mL). The chloroform solution was then precipitated twice into hexane (80 mL) and dried under reduced pressure. The CPCPA-diol product was identified by TLC (chloroform:methanol 90:10%, Rf = 0.23) and HPLC. 1 Characterized by H NMR spectroscopy (see Figure 1). Yield 3.60 g. [ka]

[0221] ii. The CPCPA-diol product from 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 minutes. DMAP (26.5 mg, 0.217 mmol) was then added and the mixture was stirred for 2 hours. 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 twice into a 9:1 MeOH / HO (v / v) solution (40 mL). The product was dissolved in chloroform (10 mL), dried over sodium sulfate, filtered, and vacuum-dried. The product was identified by TLC (chloroform:methanol 95:5%, Rf = 0.87) and NMR (MHz NMR) δ 1.01-1.025. 1 It was characterized by 1 H NMR spectroscopy. Example S2: Synthesis and characterization of dilinoleyl RAFT agents [ka]

[0222] 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 minutes. DMAP (26.5 mg, 0.217 mmol) was then added, and the mixture was stirred for 2 hours. 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 twice into a 9:1 MeOH / HO (v / v) solution (40 mL). The precipitate was collected, dissolved in chloroform (10 mL), dried over sodium sulfate, filtered, and vacuum dried. The product was purified by TLC (chloroform:methanol 95:5%, Rf = 0.87) and HCl (10 mL). 1It was characterized by 1 H NMR spectroscopy as shown in Figure 2. Example S3: Synthesis and characterization of cholesterol RAFT agents [ka]

[0223] To a sample of CPCPA (1.00 g, 3.58 mmol) in DCM (15 mL), cholesterol (1.94 g, 5.01 mmol) and DIC (2.80 mL, 17.9 mmol) were added and stirred at room temperature for 30 min. DMAP (31.6 mg, 0.259 mmol) was then added, and the mixture was stirred overnight. The solution was extracted twice with water (30 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 DCM (3 mL) and precipitated twice with a 9:1 MeOH / H2O (v / v) solution (45 mL). The mixture was centrifuged to separate the oil, which was then dissolved in DCM (3 mL) and precipitated once more with MeOH / H2O. The precipitate was collected, dissolved in DCM (10 mL), dried over sodium sulfate, filtered, and vacuum-dried. The product was identified by TLC (chloroform:methanol 95:5%, Rf=0.62) and 1 It was characterized by 1 H NMR spectroscopy as shown in Figure 3. Example S4: Synthesis of additional diacyl RAFT agents

[0224] The following diacyl RAFT agents were synthesized similarly to Examples S1-S3: [ka] Example S5: Synthesis of monoacyl RAFT agents

[0225] The following monoacyl RAFT agents were synthesized similarly to Examples S1-S3: [ka] Example S6: Synthesis of trithiocarbonate RAFT agents

[0226] The following trithiocarbonate RAFT agents were synthesized similarly to Examples S1-S3: [ka] Example S7: Synthesis of polymer-lipid compounds ("PLip") [ka]

[0227] Polymer-lipid compounds (PLips) can be synthesized using reversible addition-fragmentation chain transfer polymerization (RAFT) with acrylates or acrylamides and any one of the RAFT agents disclosed herein (e.g., Examples S1-S6) in combination with a radical initiator (e.g., 2,2'-azobis(2-methylpropionitrile), "AIBN"). The monomer(s) and RAFT agent are heated in the presence of the initiator under an inert atmosphere using a suitable solvent, such as dioxane. Grafting from the polymerization process results in growing polymer chains with the lipid tail located at the α-terminus of the polymer. One or more monomers can be incorporated into the chain to form random / statistical copolymers (monomers added together) or block / gradient copolymers (monomers added sequentially). Example S8: Synthesis of cationic PLIP (a): Synthesis of cationic diacyl PLip [ka]

[0228] i. The CPCPA-diacyl RAFT agent starting material was prepared following the general procedures disclosed in Examples S2 and S3, starting from the CPCPA-diol of Example S1.

[0229] ii. A 10 mg / mL solution of AIBN in dioxane was prepared for use in the polymerization reaction. A 2 mL sealed glass vial with a septum cap was charged with Boc-aminopropyl acrylate (55.0 mg, 0.241 mmol), the RAFT agent of Scheme S8 (24.1 mg, 0.0275 mmol), AIBN solution (0.0677 mL, 0.00413 mmol AIBN), and dioxane (0.37 mL). The flask was sealed, and the reaction mixture was sparged with nitrogen for 20 minutes before being heated to 80 °C with stirring for 8 hours. After cooling to room temperature, the solution was precipitated into hexane (14 mL) and decanted with DCM (0.2 mL). The precipitation was repeated again from 0.5 mL of DCM into hexane (14 mL). The precipitate was then dried under vacuum. 1 Analysis by H NMR and GPC: Mn 7,100 (PDI 1.26). Yield = 35-50 mg (45-65%).

[0230] iii. The dried precipitate (40 mg) from step (ii) was dissolved in 2N HCl acetic acid solution (1.0 mL) and stirred for 1 hour. Deionized water (2 mL) was added to this 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 freeze-dried. Cationic PLip was obtained. 1 It was characterized by 1 H NMR (Figure 4).

[0231] iv. The lyophilized PLip from step (iii) was dissolved in a 1:1 mixture of EtOH / HO to prepare a 20 mg / mL solution. The solution was stored at 4°C. Yield = 10-15 mg (35-60%). (b): Synthesis of cationic sterol PLip [ka]

[0232] The synthesis of cationic cholesterol PLip was carried out according to Example S8(a), substituting the diacyl RAFT agent for the cholesterol RAFT agent of Example S3. The subsequent isolation, acidification, dialysis, and formulation steps were carried out similarly to steps (ii–iv) of Example S3. Example S9: Synthesis of non-cationic PLIP [ka]

[0233] 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 placed in a 20 mL glass vial and sparged with nitrogen for 40 minutes before heating to 80 °C with stirring. After 8 hours, the solution was cooled to room temperature, and PLip was precipitated into hexane (90 mL) in three portions using ethanol as the mobile solvent (5 mL). The precipitate was dried under vacuum. 1 The product was analyzed by H NMR and GPC analysis (Figure 5). n 11,200. Yield = 801 mg (70%).

[0234] ii. The dried PLip from step (i) above was dissolved in a 1:1 mixture of EtOH / HO to prepare a 20 mg / mL solution. The solution was stored at 4°C. Example S10: Additional PLIP The following PLips were prepared according to the previous Examples S1-S9: [ka] [ka] [ka] [ka] [ka] [ka] biological example Example B1: Encapsulation of DNA into LNPs containing cationic PLIP (a) Cationic PLip incorporated into LNP formulations for DNA encapsulation

[0235] Cationic PLips 1-5, with structures shown in Table B1(a), were synthesized similarly to Synthesis Examples S1-S9. The cationic PLips were incorporated into lipid nanoparticle (LNP) formulations, and the resulting LNPs were evaluated for DNA encapsulation. Table B1(a). Structure of cationic block copolymer PLip incorporated into LNPs [Table 2] (b) Formulation of LNPs containing cationic PLip1-5

[0236] Cationic PLips 1-5 in Table B1(a) were incorporated into the corresponding LNP formulations, designated LNP formulations 1-5, respectively. Each LNP contained approximately 0.3%-1% of the given cationic PLip. As an example, the composition of LNP formulation 1 is shown in Table B1(b). LNP formulations 2-5 were prepared according to the same specifications, substituting the corresponding PLip for PLip 2-5 in each case. Table B1(b). Formulation Example 1 of LNPs containing cationic PLip1: [Table 3] (c) Encapsulation of DNA into LNPs containing cationic PLips

[0237] DNA concentration and encapsulation efficiency were measured using a modified RiboQuant assay (Thermofisher). LNPs encapsulated DNA well (73–88% encapsulation efficiency), and the presence of cationic PLip did not inhibit encapsulation (Figure 6). Example B2: PLIP-stabilized LNPs and their effect on DNA encapsulation (a) PLip incorporated into LNP formulations for DNA encapsulation

[0238] PLips 6-8, with the structures shown in Table B2(a), were synthesized similarly to Synthesis Examples S1-S9. The PLips were incorporated into lipid nanoparticle (LNP) formulations, and the resulting LNPs were evaluated for DNA encapsulation. Table B2(a). Structures of stabilized PLip6 and cationic PLip7 [Table 4] (b) Formulation of LNPs containing stabilized PLip6 and 8 and cationic PLip7

[0239] LNPs containing PLip6-8 were prepared in the mol% disclosed in Table B2(b): Table B2(b). Examples of LNP formulations containing PLip6-8: [Table 5] Encapsulation of cDNA into LNPs containing cationic PLips

[0240] 500 ng of MFP-488-labeled pDNA encapsulated in LNPs was exposed to cells for 3.5 hours at 37°C, and cell fluorescence was assessed by flow cytometry. The specific mean fluorescence intensity (SMFI), which represents the relative cell brightness compared to untreated cells, was measured as shown in Figure 7. pDNA concentration and encapsulation efficiency were measured using a modified RiboQuant assay (Thermofisher). Formulation 7 demonstrated enhanced LNP binding in two of three cell lines compared to the same LNP formulation without cationic PLip7 (shown in Figure 7). The enhanced binding was due to the cationic polymer headgroup on the PLip, which allowed ionic interactions with the cell surface that were otherwise shielded by the stabilizing uncharged lipid. (d) Size and stability of PLip-stabilized LNPs

[0241] As shown in Figure 7, stabilized PLip7 successfully stabilized LNPs in vitro and allowed them to bind to cells. Dynamic light scattering (DLS) indicated the formation of well-defined, stable LNPs (the LNPs stabilized with PLip6 had a radius of 129 nm and a PD of 9.8%). Similarly, PLip8, a PLip with poly(MSEMA) side chains, formed stable LNPs with a radius of 120 nm (PD = 17%). The formulation of LNPs containing PLip8 is disclosed in Table B2(b) above. The DLS intensity distributions of LNPs containing PLip6 and 8 are shown in Figure 8. Example B3: PLIP-stabilized LNPs and their effect on mRNA delivery in vivo (a) Synthesis and structure of stabilized PLip 9-11

[0242] Additional examples of stabilized PLips are shown below in Table B3(a): PLips 9-11 were prepared and formulated similarly to the previous examples. Table B3(a). Structures of PLip 9-11 [Table 6] (b) Formulation of LNPs containing stabilized PLip11 vs. formulation of LNPs containing PEG-DMG

[0243] Stabilized PLip11, containing a diacyl tail and a P(HPMA) head group, was used to prepare LNP formulation 11 according to Table B3(b). Reference LNPs were prepared using PEG-DMG in place of PLip11. Table B3(b). Example LNP formulation 11 containing PLip11 and reference LNP formulation containing PEG-DMG [Table 7] (c) In vivo delivery of mRNA via PLip-stabilized LNPs

[0244] Four μg of LNP-encapsulated mRNA samples were injected into four 20-25 g female Balb-c mice on days 1 and 21. Serum samples were evaluated for anti-spike protein IgG by ELISA on days 14 and 35. LNPs containing PLip11 showed a significant increase in anti-spike protein IgG by ELISA from days 14 to 35, which was comparable to LNPs made with PEG-DMG (Figure 8). Each bar represents an individual mouse. Example B4: Plasma membrane insertion of PLIP (a) Insertion of PLips bearing fluorescent functional groups into the cell membrane

[0245] The insertion of functional and / or labeled lipids into cell membranes to "prime" them has been investigated for a variety of potential applications, including the incorporation of sugars, functional groups (such as bioorthogonal "click" chemistry), and fluorescent labels into the cell surface. To demonstrate that PLips can insert into the membranes of living cells, we developed a fluorescently labeled PLip12 with the following structure: [ka] was added to Jurkat cells in either serum medium or reduced serum medium ("RSM") (Thermo Fisher Scientific Opti-MEM™ Reduced Serum Medium). Cells were incubated with increasing concentrations of fluorescent PLip12 for 1.5 hours at 37°C, followed by 60 minutes at room temperature. Cells were washed once before fluorescence assessment by flow cytometry. Cell brightness was recorded as a function of the "priming" concentration of PLip (Figure 10). The presence of serum inhibited cell priming, but priming efficiency appeared to plateau at approximately 4 μM PLip under these conditions. (b) Insertion of temperature-sensitive PLips bearing fluorescent side groups into 293F cells

[0246] Fluorescently labeled PLip13 of the following structure: [ka] Random copolymers of 1-pyrenemethyl methacrylate and NIPAm head groups were incorporated into the plasma membrane of 293F cells. To avoid endocytosis of PLip, PLip was incubated with cells at 12°C and then washed three times with phosphate-buffered saline (PBS) to remove unincorporated free PLip. Fluorescence measurements demonstrated that PLip was incorporated into cells or the plasma membrane (Figure 11). When cells were heated from room temperature to 37.5°C, the emission peak associated with pyrene dimers (approximately 480 nm) decreased compared to the emission peak associated with single pyrene monomers (approximately 396 nm). The decrease in dimer emission may be due to the local environment of pyrene; for example, when pyrene is in a hydrophobic region, it is no longer in close proximity. 293F cells (500k cells / ml in untreated 6-well plates) were pre-chilled at 12.5°C for 2 hours. Pyrene PLip was added to the cells and incubated at 12.5°C for 1 hour to promote outer membrane uptake (in the absence of endocytosis). 1 mL aliquots from each condition were washed three times with PBS. 200 μl of each condition, before and after washing, were measured on a Tecan fluorescent plate reader at room temperature and 37°C. Example B5: PLIPs with bioorthogonal reactive groups at the termini or along the backbone

[0247] We have synthesized PLips containing bioorthogonal reactive groups that can participate in chemical reactions in biological media or environments (in vivo). Examples of such bioorthogonal reactive groups include "click" chemistry groups such as strained alkynes or azide moieties. One example of a strained alkyne is the dibenzocyclooctyne (DBCO) group. (a) General reaction scheme for end-group modification of PLip [ka]

[0248] Following the aforementioned scheme, reactive PLips containing bioorthogonal DBCO functional groups were incorporated into PLips 14-19, which have the following structures: [Table 8] wherein each n is independently 0 to 100. More specifically, n is generally an integer from 1 to about 50. In certain examples disclosed herein, n has an average value of 2 to 10 (e.g., n=4.5). (b) Formulation of LNPs containing reactive / stabilized PLip14

[0249] Stabilized PLip16, containing a cholesterol lipid tail, a PEG methacrylate polymer, and DBCO functional groups, was used to form / stabilize LNPs. 1 mol% of reactive PLip was incorporated into a typical LNP mixture. An example of an LNP formulation containing a reactive PLip, such as PLip16, is LNP formulation 16 from Table B5(b). Comparison is made with the same reference formulation as Example B3(b), copied below from Table B3(b). The LNPs obtained according to formulation 16 and the reference formulation are referred to as PLip16 LNPs and PEG LNPs, respectively. Table B5(b). Exemplary LNP formulation 16 containing PLip16 and reference LNP formulation containing PEG-DMG [Table 9] (c) Conjugation of fluorescent labels to PLip-stabilized LNPs

[0250] LNPs were prepared for several PLips bearing reactive (e.g., DBCO) functional groups. The LNPs were analyzed by DLS before adding an excess of an azide-containing fluorophore (azidefluor-488 (AF488)) for 20 min. Excess AF488 was removed by dialysis. The LNPs exhibited increased fluorescence (appeared green), indicating that DBCO moieties were present in the LNPs and capable of undergoing the "click" reaction after LNP formation (Figure 12). Reference LNPs containing 0.6% PEG2k but no DBCO-PLip exhibited no fluorescence, indicating that AF488 was covalently attached to the DBCO-LNPs. Example B6: In vitro transfection reagent cationic PLip delivered alone or in a formulation (a) Effect of N:P ratio and cationic polymer length on transfection

[0251] DNA (pCILuc) complexed with various cationic PLips containing cholesterol tails and polypropylaminoacrylate head groups of 5, 13, and 19 units in length was transfected into 293F cells at various PLip / DNA ratios. The constructs are shown below. [ka]

[0252] The N:P ratio was calculated from the stoichiometry of the number of cationic amines (N) on the PLip and phosphate groups (P) on the DNA. The transfection reagent TransIT®-Jurkat (Mirus Bio) was used as a comparison. Cells were transfected with a CMV-driven firefly luciferase pDNA construct at various PLip:DNA (N:P) ratios for 48 hours. Cell harvesting was performed 48 hours posttransfection by lysing the entire well with 1% Triton-X 100 for 30 minutes at 4°C. Lysates were assayed for luciferase activity using standard conditions in a Veritas luminometer. Several cationic PLips with higher N:P ratios were shown to enhance pCILuc delivery compared to TransIT®-Jurkat. In general, higher N:P ratios increased transfection until the PLip became too toxic (Figure 13). (b) Effect of N:P ratio and cationic polymer length on the delivery of polymer / DNA complexes.

[0253] In a separate experiment, 293F cells were transfected with various DNAs (pCILuc) complexed with our proprietary polymer, POLY1, with or without cationic PLip20-22. TransIT®-Jurkat (polymer:DNA weight ratio 2:1) was used as a comparison. Cells were transfected for 48 hours with a CMV-driven firefly luciferase pDNA construct at various compound:DNA mass ratios (N:P ratios are also shown). 48 hours after transfection, cells were harvested by lysing the entire well with 1% Triton-X 100 for 30 minutes at 4°C. Lysates were assayed for luciferase activity using standard conditions on a Veritas luminometer. PLip20 did not enhance delivery of POLY1:DNA complexes. However, cationic PLips with longer polymer headgroup lengths (PLip21 and PLip22) showed significant improvements in transfection (Figure 16). Example B7: Temperature-sensitive PLIP to (de)stabilize LNPS (a) Preparation of temperature-sensitive PLips to stabilize LNPs

[0254] Poly(N-isopropylacrylamide) ("P(NIPAm)") is a polymer with a lower critical solution temperature (LCST) of 32°C. Below the LCST, the polymer is completely soluble in water (in this example, P(NIPAm) can be considered hydrophilic). Above the LCST, the polymer becomes insoluble and aggregates / precipitates due to intramolecular hydrogen bonding interactions (essentially, P(NIPAm) is considered hydrophobic). A temperature-sensitive dioleyl diglyceride, PLip (PLip23), with P(NIPAm) side chains and 55 monomer units was prepared according to the previous example; it has the following structure: [ka] (b) Incorporation of PLip18 into LNP

[0255] Three LNP formulations were prepared with varying amounts of PLip23 as disclosed in Table B6(b). Formulation 23(a) was the control and had 0 mol% PLip, while LNP formulations 23(b) and 23(c) had 0.6 mol% and 1.2 mol%, respectively. Table B7(b). Examples of LNP formulations containing PLip23 at 0, 0.6, and 1.2 mol% [Table 10]

[0256] LNPs were analyzed by DLS to determine the effect of temperature on LNP size during a cyclic temperature sweep. At room temperature (below the LCST of P(NIPAm)), LNPs with 0.6 and 1.2 mol% PLip23 were stable with a radius of 180 nm (Figure 15). LNPs with a radius of 120 nm without PLip were used as controls. When the temperature exceeded 32 °C, LNPs with PLip began to grow. The increase in radius was due to a physical change in the P(NIPAm) chain, causing lipid rearrangement. When the temperature was increased to 45 °C, LNPs containing PLip23 grew to a radius of 300 nm. When the temperature was reduced below the LCST, the LNPs shrank to their original size.

[0257] This process was shown to be reversible. As shown in Figure 16, the normalized intensity data related to particle counts did not change. These data indicate that particles did not aggregate upon the transition. Control LNPs showed no change in size or normalized intensity. Example C1: Synthesis of another temperature-sensitive "stabilized" PLIP [ka]

[0258] 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 equipped with a septum cap and a stir bar. The flask was sealed with a cap, and nitrogen was bubbled through the solution 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 with stirring. The solution was cooled to room temperature and precipitated into hexane (40 mL). The solution was reprecipitated twice more (40 mL each time) from 2.5 mL of chloroform into hexane. The collected precipitate was dried under reduced pressure. A small polymer sample was taken for NMR and GPC analysis to determine the NIPAm chain length relative to the RAFT (aromatic) end groups. NIPAm units = 115 (m = 115). Yield = 566 mg (65%).

[0259] ii) Addition of Block B (PHEA): HEA (28.0 mg, 0.241 mmol), the Block A PLip (40.0 mg, 0.00294 mmol) obtained above, AIBN solution (0.0980 mg, 0.000598 mmol AIBN), and dioxane (0.32 mL) were added to a 2 mL glass vial equipped with a septum cap and a stir bar. The flask was sealed with a cap, and nitrogen was bubbled through the solution 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 with stirring for 8 hours. The solution was cooled to room temperature and precipitated into hexane (15 mL). The solution was reprecipitated twice more (1 mL each time) from 1 mL of ethanol into hexane. The collected precipitate was dried under reduced pressure. A small polymer sample is taken for NMR and GPC analysis to determine the HEA chain length relative to block A. HEA units = 92 (n = 92). Yield = 58 mg (85%). Example D1: Stabilized complex over time

[0260] The effect of stabilized PLip (MP64240) on the functional performance of transfection complexes (e.g., VirusGEN®) over time was analyzed: percent complete capsid (Figure 17A) and genomes (Figure 17B). 293-VP 2.0 cells in virus production medium (VPM) were seeded at 3 million cells / mL immediately prior to transfection. TransIT-VirusGEN® complexes with or without temperature-sensitive PLip were formed in phosphate-buffered saline (PBS), and complex formation was measured after 30 minutes and 3.5 hours. Plasmids: pMIR699, pMIR732 AAV8, and pMIR701 AAV were harvested 72 hours post-transfection. Genomes / mL of culture fluid were measured by digital PCR (dPCR). AAV8 capsids were measured using the Lumit® AAV Capsid Immunoassay. The stabilization of VirusGEN® genome titer over 3.5 hours in the presence of temperature-sensitive PLips is shown in Figure 17B; the temperature-sensitive PLips are added to the transfection reagent in ethanol before mixing with the plasmid DNA. Example D2: Stabilization of concentrated transfection complexes

[0261] Dynamic light scattering (DLS) was used to track the aggregation behavior of transfection complexes over time. The stabilizing effect of adding a temperature-sensitive PLip (MP64240) at 5% wt / wt (relative to the lipid content in VirusGEN®) to a concentrated solution (1–5x the normal (recommended) concentration of VG and DNA in PBS) is shown in Figures 18A and 18B. The 2x and 5x concentrations indicate VirusGEN® and DNA concentrations that are 2x and 5x the recommended protocol. The presence of the temperature-sensitive PLip stabilizes transfection complexes from over 1 micron in diameter after just a few minutes (without PLip) to less than 1 micron in diameter over 2 hours (with PLip).

[0262] The functional performance of 2x and 5x VirusGEN® complexes was measured with and without a stabilizing PLip (e.g., MP64240). Figures 19A and 19B show the change in total genome titer and percent complete capsid for "regular" non-stabilized and stabilized complexes at 30 minutes and 3.5 hours. At 30 minutes, the 2x complexes exhibit similar titer and percent completeness to the standard VirusGEN (1x) complex. The 5x complexes exhibit lower titers and similar percent completeness, likely due to the larger size of the complexes at this time point. At 3.5 hours, the unstabilized 2x and 5x complexes show little functional efficacy. In contrast, when the 2x and 5x complexes are stabilized with a temperature-sensitive PLip, functional performance is maintained for longer times and at higher concentrations of complex (2x and 5x).

[0263] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein should not be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is further understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It is understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. It is therefore intended that the present invention encompass all such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be embraced therein.

Claims

1. (a) lipids; and (b) stimulus-responsive units; A compound comprising:

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

3. The polymer comprises at least three monomer units, and the at least three monomer units are C 1-20 3. The compound of claim 2, comprising a heteroalkyl side chain.

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

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

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

9. A compound comprising a lipid attached to a polymer backbone, said polymer comprising at least three monomer units, wherein said at least three monomer units are C 1-20 The compound comprises a heteroalkyl side chain.

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

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

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

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

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

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

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

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

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

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

20. 20. The compound of any one of claims 1 to 19, wherein each monomer unit is not an amino acid.

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

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

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

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

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

26. The polymer has a pK of about 2 to about 12 b 26. The compound of any one of claims 1 to 25, having the formula:

27. The polymer has a pK of about 4 to about 11. b 27. The compound of any one of claims 1 to 26, having the formula:

28. The polymer may be: 【Chemistry 1】 Contains, where: R 10 is hydrogen or C 1 ~C 6 is alkyl, and x is an integer from 1 to 20; 25. The compound of any one of claims 1 to 24.

29. Compounds according to formula I: XYZ Formula I or a pharmaceutically acceptable salt thereof; wherein: X is a lipid; Y is a polymer comprising three or more monomeric units, wherein each of said monomeric units is selected from the group consisting of C 1-20 containing a heteroalkyl side chain; and Z is an unsubstituted or substituted functional group; wherein the lipid is covalently attached to the polymer via the backbone of the polymer.

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

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

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

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

34. 31. 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. 35. The compound of claim 30 or claim 34, wherein the fatty acid comprises oleic acid or an ester thereof.

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

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

38. Lipids have an octanol:water coefficient (log(K)) of about 2 or greater. OW ) ...

39. The compound of any one of claims 1 to 38, wherein the lipid has a structure of Formula XA, Formula XB, or Formula XC: 【Chemistry 2】

40. 40. The compound of any one of claims 1 to 39, wherein the polymer has a structure of formula YA, formula YB, formula YC, or formula YD: 【Transformation 3】 or a pharmaceutically acceptable salt thereof; A 1 , B 1 , C 1 , and D 1 each is independently hydrogen or methyl; A 2 , B 2 , C 2 , and D 2 each independently represents an unsubstituted or substituted C 1-20 is heteroalkyl; 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 greater; Here, the substitution C 1-20 each heteroalkyl 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; The compound.

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

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

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

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

45. A 2 , B 2 , C 2 , and D 2 45. The compound of any one of claims 29-44, wherein each of independently comprises an acrylate or an acrylamide.

46. 46. ​​The compound of any one of claims 3 to 45, wherein each monomer unit has a structure of any one of the following formulae: 【Chemistry 4】 or a pharmaceutically acceptable salt thereof; R 1 and R 3 each is independently hydrogen or methyl; Each R 2 is hydrogen, C 1-6 Alkyl, C 7-20 Aralkyl, C 1-20 heteroalkyl, or a polyethylene glycol chain containing 1 to 100 ethylene glycol monomers; 1-6 Alkyl, C 7-20 Aralkyl, and C 1-20 Each heteroalkyl is unsubstituted or substituted with one or more groups, wherein each of said one or more groups is independently selected from the group consisting of COOH, -CONH 2 , -NH 2 , -NH 3 + , -NHC(NH 2 + )NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -N(CH 3 ) 3 + , -OH, -OCH 3 , -SH, -S(O)CH 3 , -S(O) 2 CH 3 , or -S(O) 2 OH; R 4 , and R 5 each independently represents 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 heteroalkyl is unsubstituted or substituted with one or more groups, wherein each of said one or more groups is independently selected from the group consisting of COOH, -CONH 2 , -NH 2 , -NH 3 + , -NHC(NH 2 + )NH 2 , -NHCH 3 , -N(CH 3 ) 2 , -N(CH 3 ) 3 + , -OH, -OCH 3 , -SH, -S(O)CH 3 , -S(O) 2 CH 3 , or -S(O) 2 OH; or a pharmaceutically acceptable salt thereof. The compound.

47. The compound of any one of claims 3 to 46, wherein each monomer unit independently comprises: 【Transformation 5】

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

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

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

51. 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. Sulfide is SR 6 and where R 6 51. The compound of claim 50, wherein comprises a reactive group, a charged group, a detectable group, a peptide group, a capping group, or a combination thereof.

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

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

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

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

58. 57. The compound of any one of claims 28 to 56, wherein the functional group is: 【Transformation 6】 Selected from; wherein the functional group is attached to the polymer via a sulfur atom.

59. 59. The compound of any one of claims 1 to 58, wherein the compound is configured to encapsulate or complex nucleic acids in aqueous solution.

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

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

62. 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. 63. A nanoparticle comprising a compound according to any one of claims 1 to 62 and configured for encapsulation or complexation of nucleic acids.

64. 64. The nanoparticle of claim 63, wherein the nanoparticle is configured to encapsulate or complex the nucleic acid at a ratio of 0.3:1 to 100:1 (weight:weight).

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

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

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

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

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

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

71. 64. A transfection reagent comprising the nanoparticles of claim 63 and nucleic acid encapsulated therein.

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

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

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

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

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

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

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

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

81. 1. A method for transfecting a cell, 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 said transfection reagent, wherein said contacting is performed under conditions suitable for entry of said nucleic acid into said cell.

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

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

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

86. 86. The method of any one of claims 81 to 85, wherein the contact is for less than 24 hours.

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

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

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

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

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

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

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

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

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

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

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

98. 90. 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. 99. The method of claim 98, wherein the pharmaceutical composition is administered to the subject by injection.

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