Polyoxazoline-lipid conjugates, lipid nanoparticles and pharmaceutical compositions containing the same

Abstract

POZ-lipid conjugates and lipid nanoparticles (LNPs) comprising POZ-lipid conjugates are used to facilitate delivery of encapsulated payloads. LNPs comprising POZ-lipid conjugates and nucleic acid payloads, such as but not limited to mRNA or modified mRNA, are disclosed. Such LNPs are not immunogenic or have reduced immunogenicity compared to the corresponding LNPs comprising PEG-lipids.

Description

[Technical field]

[0001] The present disclosure relates to polyoxazoline-lipid conjugates, methods for the synthesis of these conjugates, and their use in lipid nanoparticles and pharmaceutical compositions. Lipid nanoparticles incorporating oligonucleotides, such as mRNA, DNA, and siRNA, for delivery to living cells are also contemplated. [Background technology]

[0002] Nucleic acid (especially mRNA) based vaccines offer several advantages over other vaccine technologies. For example, nucleic acid based vaccines can be rapidly produced using manufacturing platforms and purification methods common regardless of the oligonucleotide payload, reducing development time and cost. Furthermore, mRNA based vaccines may be able to present polypeptides as antigens when taken up by cells capable of translating the encoded protein. These cells include antigen presenting cells such as macrophages and dendritic cells.

[0003] However, current nucleic acid-based vaccines have several drawbacks. For example, mRNA is rapidly degraded by nucleases in the body and is not easily taken up by most cell types when only complexed to polyamines such as protamine. Moreover, a major factor hindering the development of both DNA and mRNA vaccines is the lack of a potent, well-tolerated, non-immunogenic delivery system that can be administered repeatedly without serious adverse events.

[0004] In an attempt to address these shortcomings, mRNA payloads (and other oligonucleotide payloads) have been encapsulated into lipid nanoparticles (LNPs), thereby protecting the mRNA from enzymatic degradation and enhancing cellular uptake and expression up to 1000-fold compared to mRNA complexed to polyamines. Such LNPs are typically composed of an ionizable lipid (to complex with the oligonucleotide), cholesterol (to provide flexibility), lipids containing polyethylene glycol (PEG) moieties (to stabilize the lipid nanoparticle and prevent fusion with other nanoparticles), and helper lipids such as distearoylphosphatidylcholine (DSPC) (to provide structural integrity). For example, US Patent Publication No. 2020 / 0230058 discloses liposomes in which RNA encoding an immunogen of interest is encapsulated, the liposomes comprising at least one PEG-lipid, the PEG being present on the exterior of the liposome, and having an average molecular mass of 1 kDa to 3 kDa.

[0005] Early studies working with small interfering RNA (siRNA) identified ionizable lipids as one of the key components essential for efficacy. Ionizable lipids are crucial for endosomal escape once LNPs become trafficked through the endosomal compartment in cells.

[0006] LNPs are generally considered to be biocompatible nanocarriers with acceptable safety profiles and capabilities to carry oligonucleotide payloads. Nevertheless, as briefly mentioned above, problems have been recognized regarding the immunogenicity of some LNPs when administered to animals and humans. In particular, PEG-lipids, which are often used in LNPs, may compromise vaccine safety due to the effects of anti-PEG immune responses. In fact, it is increasingly recognized that treating patients with PEGylated components, including PEG-lipids, may lead to the formation of antibodies that specifically recognize and bind to PEG (i.e., anti-PEG antibodies). Moreover, anti-PEG antibodies have been found in patients who have not been treated with PEGylated drugs but have been exposed to products containing PEG (e.g., cosmetics and foods).

[0007] Thus, treating patients with preformed anti-PEG antibodies with LNPs containing PEG-lipids may accelerate blood clearance of the LNPs containing PEG-lipids, reduce / impair efficacy, and cause hypersensitivity and possibly severe allergic reactions to PEG. This immunogenicity of PEG may cause serious adverse events if subjects receive repeated vaccinations with LNPs containing PEG-lipids over time or if they have been previously exposed to products containing PEG. Indeed, in the early ramp-up of mRNA vaccinations from Pfizer / BioNTech and Moderna worldwide against SARS-CoV-2 ("COVID-19"), caregivers reported an unusually high incidence of life-threatening adverse reactions, including anaphylaxis. The majority of anaphylaxis occurred in women (approximately 90%) and was shown to be due to antibodies against PEG. Although the exact mechanism mediating anaphylaxis is still unknown, the life-threatening immune response is likely due to basophil degranulation in patients previously sensitized to PEG through exposure to PEG-containing cosmetics, foods, or medicines. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 2020 / 0230058 Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there remains a need to address the shortcomings of current LNP technology and improve methods for delivering nucleic acid vaccines. In particular, it would be advantageous to identify LNP formulations with significantly reduced or absent immunogenicity. It would also be advantageous to identify LNP formulations with acceptable reactogenicity profiles after initial and subsequent administrations of LNP. Furthermore, while the role of the intracellular stability of the polymer-lipid in LNP formulations has not yet been fully explored and the details of cellular uptake and subsequent release of oligonucleotides into cells are still poorly understood, it is generally believed (although this point is controversial) that the polymer-lipid in LNPs must be shed from the LNP in order for the payload to be released into the cell. For these and other reasons, it is essential in the field of LNP technology to have reduced or non-immunogenic polymer lipids available to provide a range of in vivo stability. The POZ-lipid conjugates of the present disclosure provide a reduced or non-immunogenic alternative to the PEG-lipids used in current LNP formulations, and thus also provide improved non-immunogenic LNPs for use in vaccine delivery systems. Additionally, LNPs produced according to the present disclosure possess several properties that are potentially beneficial to the art, including, but not limited to, particle size, polydispersity, freeze / thaw stability, oligonucleotide encapsulation efficiency, maintenance of oligonucleotide integrity, endosomal escape, transfection efficiency, and the like. [Means for solving the problem]

[0010] Summary of the Invention The present disclosure relates to a novel polymer lipid comprising polyoxazoline (POZ) bound to lipid, and having variable stability between the polymer and the lipid in biological system.In this embodiment, polyoxazoline-lipid (POZ-lipid) conjugate has controllable degradability through the linkage between POZ and lipid.POZ-lipid conjugate can be incorporated into lipid nanoparticles, and in so doing, can impart unique properties to lipid nanoparticles.

[0011] In one embodiment, the lipid nanoparticle composition of the present disclosure can include a POZ-lipid as described herein. In another embodiment, the composition of the present disclosure can include a POZ-lipid as described herein, a cationic or ionizable lipid, and an optional additional lipid. In yet another embodiment, the composition of the present disclosure can include a POZ-lipid as described herein, an additional lipid, such as a phospholipid or structural lipid, cholesterol, an oligonucleotide payload, and combinations thereof. The lipid nanoparticle composition that encapsulates the oligonucleotide payload can result in expression of the payload in a suitable cell type that takes up the lipid nanoparticle, thus resulting in a therapeutic response to the payload. In one embodiment, the oligonucleotide payload includes, but is not limited to, an mRNA vaccine against infectious diseases such as SARS-CoV-2, rabies, and influenza. In another embodiment, the lipid nanoparticle composition can also be used in a variety of therapeutic procedures, including, but not limited to, cancer immunotherapy, gene therapy, enzyme replacement, and combinations thereof.

[0012] The present disclosure also provides compounds of formula I: R-POZ-L-lipid I Also, in the formula: R comprises an initiator group; POZ comprises a polyoxazoline polymer; L comprises a linking group that is controllably degradable in a physiological medium; The lipids include uncharged lipids that contain at least one hydrophobic moiety.

[0013] In one embodiment, POZ in formula I is -{[N(COX)CH2CH2] o -[N(COR2)CH2CH2] n} a - and where X comprises a functional group including an alkyne group, a triazole having an attached carboxylic acid, or a combination thereof; o ranges from 0 to 10; n ranges from 1 to 1000; R2 comprises hydrogen, a substituted or unsubstituted alkyl, an alkyne substituted alkyl, or a substituted or unsubstituted aralkyl group; and a is ran, which represents a random copolymer, or block, which represents a block copolymer.

[0014] In another embodiment, the POZ has a polydispersity index of about 1.01 to about 1.20. In yet another embodiment, the POZ has a molecular weight of 500 Daltons to 5,000 Daltons. In yet another embodiment, R includes hydrogen, substituted or unsubstituted alkyl, alkyne-substituted alkyl, triazole with an attached carboxylic acid, or substituted or unsubstituted aralkyl group. L can include ethers, esters, carboxylates, carbonates, carbamates (including but not limited to ethyl carbamate (urethane)), amines, amides, disulfides, and combinations thereof. In another embodiment, the lipid can include two hydrophobic moieties. For example, the lipid can include a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof. In one embodiment, the lipid includes 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

[0015] In this aspect of the disclosure, Formula I can be one of the following:

[0016] [ka] TIFF2024505723000003.tif158165TIFF2024505723000004.tif143166In the formula, m is 1 to 2, n is in the range of 1 to 1000, o is in the range of 1 to 5, and p is in the range of 1 to 10. The present disclosure also provides a compound of formula II Lipid-L1-(POZ) n a -T II Also, in the formula: The lipid comprises an uncharged lipid comprising at least one hydrophobic moiety; L1 comprises a linking group that is controllably degradable in a physiological medium; POZ comprises a polyoxazoline polymer of the structure [N(COR2)CH2CH2], where R2 is independently selected from unsubstituted or substituted alkyl, alkenyl, alkyne-substituted alkyl, aralkyl, heterocyclylalkyl, or an active functional group for each repeat unit of the polyoxazoline polymer; n is in the range of 1 to 1,000; a is ran representing a random copolymer or block representing a block copolymer; T includes a terminating end group.

[0017] In this aspect, L1 can include ethers, esters, carboxylates, carbonates, carbamates (including but not limited to ethyl carbamate (urethane)), amines, amides, disulfides, and combinations thereof. In one embodiment, L1 is a triazole. In another embodiment, T comprises ZBQ, where Z comprises S, O, or N, B is an optional linking group, and Q is a terminating nucleophile or a portion thereof.

[0018] In this aspect, the lipid can include two hydrophobic moieties.For example, the lipid can include a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof.In one embodiment, the lipid includes 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

[0019] In one embodiment, Formula II is one of the following:

[0020] [ka] TIFF2024505723000006.tif199166TIFF2024505723000007.tif43165In the formula, m is 1 to 2, n is in the range of 1 to 1000, and o is in the range of 1 to 5.

[0021] The present disclosure also provides a compound of formula III R-(POZ) n a -Z-L2-lipid III Also, in the formula: R comprises an initiator group; POZ comprises a polyoxazoline polymer of the structure [N(COR2)CH2CH2], where R2 is independently selected for each repeat unit of the polyoxazoline polymer from an unsubstituted or substituted alkyl, alkenyl, alkyne-substituted alkyl, aralkyl, heterocyclylalkyl group, or an active functional group; n is in the range of 1 to 1,000; a is ran representing a random copolymer or block representing a block copolymer; Z comprises S, O, or N; L2 comprises a linking group that is controllably degradable in a physiological medium; The lipids include uncharged lipids that contain at least one hydrophobic group.

[0022] In this aspect, L2 can include ethers, esters, carboxylates, carbonates, carbamates (including but not limited to ethyl carbamate (urethane)), amines, amides, disulfides, and combinations thereof. In one embodiment, the lipid includes two hydrophobic moieties. For example, the lipid can include a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof. In one embodiment, the lipid includes 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

[0023] In one embodiment, R comprises hydrogen or substituted or unsubstituted alkyl and n is in the range of 15-35.

[0024] The present disclosure also provides a compound of formula IV

[0025] [ka] Also, in the formula: R comprises an initiator group; L3 comprises a linking group that has controllably degradability in a physiological medium; The lipid comprises an uncharged lipid comprising at least one hydrophobic moiety; n is in the range of 1 to 5; R2 is independently selected for each repeat unit from unsubstituted or substituted alkyl, alkenyl, alkyne-substituted alkyl, aralkyl, heterocyclylalkyl, or an active functional group; m is in the range of 1 to 100; a is ran representing a random copolymer or block representing a block copolymer; T includes a terminating end group.

[0026] In this aspect, L3 can include ethers, esters, carboxylates, carbonates, carbamates, amines, amides, disulfides, and combinations thereof. In one embodiment, L3 includes a triazole. In another embodiment, T includes ZBQ, where Z includes S, O, or N, B is an optional linking group, and Q is a terminating nucleophile or a portion thereof.

[0027] The lipid can include two hydrophobic moieties. For example, the lipid can include a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof. In one embodiment, the lipid can include 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

[0028] In one embodiment, any of the compounds of Formulas I-IV above have a hydrolysis rate determined at least in part by L, L1, L2, or L3 (as applicable). In another embodiment, any of the compounds of Formulas I-IV above have a half-life for hydrolysis in 50% human plasma of about 10 minutes or less. In yet another embodiment, any of the compounds of Formulas I-IV above have a half-life for hydrolysis in 50% human plasma of about 120 hours or more.

[0029] A composition may be formed comprising any of the compounds of formula I-IV above. In one embodiment, such a composition may further comprise a cationic or ionizable lipid. In another embodiment, the present disclosure relates to a method for treating a disorder or disease in an animal, comprising administering to the animal an effective amount of such a composition. In yet another embodiment, the present disclosure relates to a method for eliciting a protective immune response in an animal, comprising administering to the animal an effective amount of such a composition. In either case, the administering step may comprise delivering such a composition to the animal via a subcutaneous, intravenous, intramuscular, intradermal or aerosol route.

[0030] Other features and advantages of the present invention can be seen from the following detailed description taken in conjunction with the drawings described below. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 illustrates a lipid nanoparticle according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present disclosure provides various novel POZ-lipid conjugates of the present disclosure and lipid nanoparticles (LNPs) comprising the POZ-lipid conjugates. The LNPs may be used to facilitate intracellular delivery of bioactive therapeutic molecules. In one embodiment, the LNPs may be used to deliver encapsulated payloads, such as nucleic acid payloads, including but not limited to mRNA or modified mRNA. Since LNPs comprising the POZ-lipid conjugates of the present disclosure are non-immunogenic or have reduced immunogenicity compared to corresponding LNPs comprising PEG-lipids, such LNPs provide a safer method of delivering nucleic acid vaccines. Furthermore, without being bound to a particular theory, it is expected that the novel POZ-lipids, as components of LNPs, may also impart unique properties compared to PEG-lipids, and the resulting LNPs may exhibit unique uptake, distribution and efficacy when administered as therapeutics. Furthermore, the POZ-lipids of the present disclosure may advantageously have varying degrees of stability, providing additional flexibility in "engineering" LNPs comprising POZ-lipids. The present disclosure also relates to pharmaceutical compositions comprising such LNPs, which are useful for delivering a therapeutically effective amount of a bioactive molecule to cells in a patient.

[0033] Polyoxazolines (POZ) are biocompatible polymers that retain good solubility in many hydrophilic and hydrophobic solvents. POZ are also resistant to oxidative degradation and do not undergo bioaccumulation. POZ imparts stealth capability and good mucosal tissue penetration when grafted to silica particles, gold particles, ZnO nanocrystals, and magnetic particles.

[0034] LNPs are amphiphilic spherical vesicles with sizes ranging from about 20 nm to several microns, formed by one or more lipid layers enveloping an aqueous core. Prior art LNPs generally contain cationic or ionizable lipids in combination with (i) helper lipids to support the bilayer structure and promote endocytosis; (ii) sterol lipids (i.e., cholesterol) to stabilize the lipid bilayer of the LNP; and (iii) PEG-lipids to provide LNPs with hydratable layers to improve colloidal stability, prevent fusion of nascent particles, reduce protein adsorption and nonspecific uptake, and prevent reticuloendothelial clearance. However, as described above, the PEG-lipids in these LNPs may compromise patient safety due to the possibility of anti-PEG immune responses. The use of the POZ-lipids of the present disclosure, which have never been used in LNPs, particularly LNPs for nucleic acid delivery, provides a solution to the immunogenicity problem of PEGylated LNPs, and also provides LNPs with unique uptake, distribution, and efficacy (compared to PEGylated LNPs) when administered as therapeutics.

[0035] definition All patent applications, patents, and printed publications cited herein are incorporated herein by reference in their entirety, except to the extent that any definitions, disclaimers or denials of subject matter, and the incorporated material is inconsistent with an explicit disclosure in this specification, in which case the language in this disclosure will control.

[0036] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0037] The terms "about" and "approximately" are intended to mean an acceptable range of error or variation in the quantity measured, generally given the nature or precision of the measurements. Numerical quantities set forth herein are approximate unless otherwise stated, and the term "about" or "approximately" means that they can be inferred when not expressly stated.

[0038] As used herein, the terms "active" or "activated" when used in reference to a particular functional group, refers to a functional group that readily reacts with an electrophile or nucleophile of another molecule. This is in contrast to groups that require a catalyst or impractical reaction conditions in order to react (i.e., "nonreactive" or "inactive" groups).

[0039] As used herein, the term "physiologically degradable" or "physiologically releasable" refers to a linkage that includes a cleavable moiety. The terms degradable and releasable do not imply any particular mechanism by which the linker is cleaved.

[0040] As used herein, the terms "link," "linked," "linkage," or "linker," when used with respect to a POZ polymer, a POZ conjugate, an agent, or a compound described herein, or a component thereof, refer to a bond that is usually formed as a result of a chemical reaction, and is typically a covalent bond.

[0041] The term "lipid nanoparticle" or "LNP" is used herein to encompass any of many types of nanoparticles, including liposomes, formed by lipid layers surrounding a core containing molecules to be delivered to the body. Liposomes generally have one or more contiguous lipid bilayers encapsulating an aqueous core. Other forms of liposome-like nanocarriers may have lipid monolayers or non-contiguous bilayers, and may or may not have an aqueous core.

[0042] The term "hydrophilic" as used herein refers to compounds or molecules, or portions thereof, in which interactions with water are thermodynamically more favorable than interactions with oil or other hydrophobic solvents, e.g., with respect to hydrophilic groups. Hydrophilic compounds can be dissolved or dispersed in water.

[0043] The term "hydrophobic" as used herein, for example with respect to hydrophobic moieties, refers to compounds or molecules, or portions thereof, in which interaction with water is thermodynamically less favorable than interaction with oil or other hydrophobic solvents. Hydrophobic compounds can be dissolved or dispersed in oil or other hydrophobic solvents.

[0044] As used herein, the terms "inert" or "non-reactive" when used in reference to a particular functional group refers to a functional group that does not readily react with electrophiles or nucleophiles of another molecule, requiring a catalyst or impractical reaction conditions in order to react.

[0045] As used herein, the term "pendent group" refers to a portion of a POZ polymer that is attached to the POZ polymer.

[0046] The term "pendent moiety," as used herein, refers to a substituent that is linked to a POZ polymer moiety via a linking group. A pendent moiety is exemplified by R2 of formula IV described herein.

[0047] As used herein, the term "pharmaceutically acceptable" refers to a compound that is compatible with the other components of a composition and is not harmful to the subject receiving the compound or composition. In some embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly, in humans.

[0048] As used herein, the term "pharmaceutically acceptable form" is intended to include known forms of a compound or POZ conjugate that may be administered to a subject, including, but not limited to, solvates, hydrates, prodrugs, isomorphs, polymorphs, pseudomorphs, neutral forms, and salt forms of the compound. In some embodiments, pharmaceutically acceptable forms exclude prodrugs, isomorphs, and / or pseudomorphs. In some embodiments, pharmaceutically acceptable forms are limited to pharmaceutically acceptable salts, neutral forms, solvates, and hydrates. In some embodiments, pharmaceutically acceptable forms are limited to pharmaceutically acceptable salts and neutral forms. In some embodiments, pharmaceutically acceptable forms are limited to pharmaceutically acceptable salts.

[0049] The term "alkyl," as used herein, whether used alone or as part of a substituent group, is a term of art that refers to saturated aliphatic groups that optionally contain one or more heteroatoms (such as O, S, or N), which may be optionally substituted, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In some embodiments, a straight chain or branched chain alkyl group has about 30 or less carbon atoms in its backbone (e.g., C1-C for straight chain). 30 , C3~C for branched chains 30 ), or about 20 or less, or 10 or less carbon atoms. In some embodiments, the term "alkyl" refers to any of C1 to C6 10 In some embodiments, the term "alkyl" refers to a C3 to C4 alkyl group. 12 In some embodiments, the term "alkyl" refers to a C3-C8 branched alkyl group. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, and n-hexyl. In some embodiments, the term "alkyl" refers to a C1-C8 branched alkyl group, including one or more heteroatoms (such as O, S, or N) that may be optionally substituted in place of a carbon atom.10 In some embodiments, the term "alkyl" refers to a C1-C6 alkyl group substituted with up to five groups selected from the group consisting of OH, NH2, and =O. 10 Refers to a straight chain alkyl group.

[0050] The term "alkenyl" as used herein, whether used alone or as part of a substituent group, is a term of art and refers to an unsaturated aliphatic group, optionally containing one or more heteroatoms (such as O, S or N), which may be optionally substituted, including straight or branched chain hydrocarbon groups containing 2-30 carbons and at least one carbon-carbon double bond formed by removing two hydrogens. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butynyl, 4-pentynyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl. The unsaturated bond of the alkenyl group may be located anywhere in the moiety and may have a (Z) or (E) configuration about the double bond.

[0051] The term "alkynyl," as used herein, whether used alone or as part of a substituent group, is a term of art that refers to an unsaturated aliphatic group containing a straight or branched chain hydrocarbon group containing 2 to 30 carbon atoms and at least one carbon-carbon triple bond, optionally containing one or more heteroatoms (such as O, S, or N), which may be optionally substituted. Representative examples of alkynyl include, but are not limited to, acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 4-pentynyl, and 1-butynyl.

[0052] As used herein, the terms "substituted alkyl," "substituted alkenyl," and "substituted alkynyl" refer to groups in which one or more bonds to a carbon or hydrogen has been replaced with a bond to a non-hydrogen or non-carbon atom, for example, but not limited to, a halogen atom in a halide such as F, Cl, Br, or I; an oxygen atom in a group such as a carbonyl, carboxyl, hydroxyl, alkoxy, aryloxy, heterocyclyloxy, or ester group; a sulfur atom in a group such as a thiol, alkyl and aryl sulfide, sulfone, sulfonyl, or sulfoxide group; an amine, amide, alkylamine, dialkylamine, aryl ... The term "polar alkyl", "polar alkenyl", and "polar alkynyl" refers to alkyl, alkenyl, and alkynyl groups as defined above, which are replaced by bonds to nitrogen atoms in groups such as alkylarylamines, diarylamines, N-oxides, imides, enamines, imines, oximes, hydrazones, heterocyclylamines, (alkyl)(heterocyclyl)-amines, (aryl)(heterocyclyl)amines, diheterocyclylamines, triazoles, nitriles, and the like; silicon atoms in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and other heteroatoms in various other groups. In specific embodiments, "polar alkyl", "polar alkenyl", and "polar alkynyl" refer to alkyl, alkenyl, and alkynyl groups substituted with atoms that form polar covalent bonds. In another specific embodiment, "polar alkyl", "polar alkenyl", and "polar alkynyl" refer to C1-C5 alkyl, alkenyl, and alkynyl groups substituted with atoms that form polar covalent bonds. In specific embodiments, "polar alkyl", "polar alkenyl", and "polar alkynyl" refer to alkyl, alkenyl, and alkynyl groups, such as C1-C5 alkyl, alkenyl, and alkynyl groups, substituted with -OH and / or -C(O)-OH groups.

[0053] The terms "halo" or "halogen," as used herein, whether used alone or as part of a substituent, are terms of art and refer to -F, -Cl, -Br, or -I.

[0054] The term "alkoxy," as used herein, whether used alone or as part of a substituent group, is a term of art and refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0055] The term "aralkyl" or "arylalkyl" as used herein, whether used alone or as part of a substituent, is a term of art and refers to an alkyl group substituted with an aryl group, which is attached to the parent molecule through the alkyl group. The arylalkyl group may be optionally substituted. "Substituted aralkyl" has the same meaning with respect to unsubstituted aralkyl groups that substituted aryl groups had with respect to unsubstituted aryl groups. However, substituted aralkyl groups also include groups in which a carbon or hydrogen bond of the alkyl moiety is replaced by a bond to a non-carbon or non-hydrogen atom.

[0056] As used herein, the term "heteroaralkyl" or "heteroarylalkyl", whether used alone or as part of a substituent group, is a term of art that refers to an alkyl group substituted with a heteroaryl group, which is attached to the parent molecular moiety through the alkyl group. The heteroarylalkyl may be optionally substituted. The term "substituted heteroarylalkyl" has the same meaning with respect to unsubstituted heteroarylalkyl groups that substituted aryl groups had with respect to unsubstituted aryl groups.

[0057] The term "heterocyclylalkyl" as used herein, whether used alone or as part of a substituent, is a term of art and refers to an unsubstituted or substituted alkyl, alkenyl, or alkynyl group in which a hydrogen or carbon bond of the unsubstituted or substituted alkyl, alkenyl, or alkynyl group is replaced with a bond to a heterocyclyl group. A heterocyclylalkyl may be optionally substituted. The term "substituted heterocyclylalkyl" has the same meaning with respect to unsubstituted heterocyclylalkyl groups that substituted aryl groups had with respect to unsubstituted aryl groups. However, substituted heterocyclylalkyl groups also include groups in which a non-hydrogen atom is bonded to a heteroatom in the heterocyclyl group of the heterocyclylalkyl group, such as, but not limited to, a nitrogen atom in the piperidine ring of a piperidinylalkyl group.

[0058] The term "aryl" as used herein, whether used alone or as part of a substituent, is a term of art and refers to monocyclic, bicyclic and polycyclic aromatic hydrocarbon groups, such as benzene, naphthalene, anthracene and pyrene. The aromatic ring may be substituted at one or more positions of the ring with one or more substituents, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moiety, fluoroalkyl (such as trifluromethyl), cyano. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are shared between two adjacent rings (the rings are "fused rings"), where at least one of the rings is aromatic hydrocarbon, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. In some embodiments, the term "aryl" refers to a phenyl group. The aryl group may be optionally substituted.

[0059] The term "cycloalkyl," as used herein, whether used alone or as part of a substituent, is a term of art that refers to a saturated carbocyclic group containing from 3 to 6 ring carbon atoms, such rings being optionally substituted with substituted or unsubstituted alkyl groups or the substituents described for substituted alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-methylcyclobutyl, and 4-ethylcyclohexyl.

[0060] The term "heteroaryl," as used herein, whether used alone or as part of a substituent group, is a term of art that refers to monocyclic, bicyclic, and polycyclic aromatic groups having a total of 3 to 30 atoms in the ring structure, including one or more heteroatoms, such as nitrogen, oxygen, or sulfur. Exemplary heteroaryl groups include azaindolyl, benzo(b)thienyl, benzimidazolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzoxadiazolyl, furanyl, imidazolyl, imidazopyridinyl, indolyl, indolinyl, indazolyl, isoindolinyl, isoxazolyl, isothiazolyl, isoquinolinyl, oxadiazolyl, oxazolyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrrolyl, pyrrolo[2,3-d]pyrimidinyl, pyrazolo[3,4-d]pyrimidinyl, quinolinyl, quinazolinyl, triazolyl, thiazolyl, thiophenyl, tetrahydroindolyl, tetrazolyl, thiadiazolyl, thienyl, thiomorpholinyl, triazolyl or tropanyl, and the like. A "heteroaryl" may be substituted at one or more ring positions with one or more substituents, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amide, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, fluoroalkyl (such as trifluoromethyl), cyano, etc. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are shared between two adjacent rings (the rings are "fused rings"), where at least one of the rings is an aromatic group having one or more heteroatoms in the ring structure, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl.

[0061] As used herein, the term "heterocyclyl", whether used alone or as part of a substituent group, is a term of art which may be fully saturated or may contain one or more units of unsaturation, but for the avoidance of doubt, the degree of unsaturation does not result in an aromatic ring system and refers to groups of non-aromatic ring systems, including but not limited to monocyclic, bicyclic, and tricyclic rings, having 3-15 atoms including at least one heteroatom, such as nitrogen, oxygen or sulfur. The following are given by way of example and should not be construed as limiting the scope of the invention: aziridinyl, azirinyl, oxiranyl, thiiranyl, thiirenyl, dioxiranyl, diazirinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, azetyl, oxetanyl, oxetyl, thietanyl, thiethyl, diazetidinyl, dioxetanyl, dioxetenyl, dithietanyl, dithiethyl, dioxalanyl, oxazolyl, thiazolyl, triazinyl, isothiazolyl, Isoxazolyl, azepine, azetidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxopiperidinyl, oxopyrrolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, quinuclidinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl are examples of heterocyclic rings.Heterocyclyl groups may be substituted at one or more ring positions with one or more substituents, such as halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, fluoroalkyl (such as trifluoromethyl), cyano, and the like.

[0062] As used herein, the terms "treatment," "treat," and "treating" refer to a course of action (such as administering a conjugate described herein or a pharmaceutical composition comprising a conjugate described herein) to prevent, eliminate, or reduce a symptom, aspect, or characteristic of a disease or condition. Such treatment need not be absolute to be useful. In one embodiment, treatment includes a course of action initiated simultaneously with or following the onset of a symptom, aspect, or characteristic of a disease or condition. In one embodiment, treatment includes a course of action initiated prior to the onset of a symptom, aspect, or characteristic of a disease or condition.

[0063] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver that a patient requires or would benefit from treatment, which judgment is based on a variety of factors that are within the caregiver's expertise, but includes knowledge that the patient is or will be ill as a result of a disease or condition treatable by the methods or compounds of the present disclosure.

[0064] The terms "individual", "subject", or "patient" as used herein refer to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, and humans. Terms can specify male or female, or both, or can exclude male or female. In preferred embodiments, the terms "individual", "subject", or "patient" refer to humans.

[0065] As used herein, the term "therapeutically effective amount" refers to an amount of a conjugate alone, or as part of a pharmaceutical composition, that is capable of having any detectable positive effect on any symptom, aspect, or characteristic of a disease or condition. Such an effect need not be absolute to be beneficial.

[0066] It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is subject to the permissible valence of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation, such as by rearrangement, fragmentation, decomposition, cyclization, elimination, or other reaction.

[0067] When a group is specified as a moiety of a compound, it is understood that the substitution of the group can be adjusted to accommodate specific bonds. For example, when an alkyl group is linked to two other groups, the alkyl group is considered an alkylene group.

[0068] The term "substituted" is also intended to encompass all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclyl, aromatic and nonaromatic substituents of organic compounds. Exemplary substituents include, for example, those described herein above. In this disclosure, heteroatoms such as oxygen or nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. Exemplary substitutions include, but are not limited to, hydroxy, halogen, azido, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, fluoroalkyl (such as trifluoromethyl), cyano, and the like. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

[0069] Other chemical terms herein are used according to conventional usage in the art as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., ed., 1985, McGraw-Hill, San Francisco), which is incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0070] As used herein, the term "pharmaceutically acceptable salt" includes salts derived from inorganic or organic acids, including, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, phosphoric acid, formic acid, acetic acid, lactic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, glycolic acid, salicylic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, malonic acid, trifluoroacetic acid, trichloroacetic acid, naphthalene-2-sulfonic acid, and other acids. Pharmaceutically acceptable salt forms can include forms in which the ratio of salt-containing molecules is not 1:1. For example, the salt can include more than one molecule of inorganic or organic acid per molecule of base, such as two molecules of hydrochloric acid per molecule of conjugate. As another example, the salt can include less than one molecule of inorganic or organic acid per molecule of base, such as two molecules of conjugate per molecule of inorganic or organic acid.

[0071] As used herein, the terms "carrier" and "pharmaceutical acceptable carrier" refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered or formulated for administration. Non-limiting examples of such pharmaceutical acceptable carriers include liquids such as water, saline, and oils, and solids such as gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, and urea. Additionally, auxiliary agents, stabilizers, thickeners, iso-osmotic agents, cryopreservatives, lubricants, flavoring agents, and coloring agents may be used. Other examples of suitable pharmaceutical carriers are described in Remington's Science and Practice of Pharmacy (23rd Edition, ISBN 9780128200070) and Handbook of Pharmaceutical Excipients (8th Edition, 978-0-85-711271-2), each of which is incorporated herein by reference in its entirety.

[0072] As used herein, the term "targeting molecule" refers to any molecule having a therapeutic or diagnostic application or targeting function, or vehicle with which a compound is administered or formulated for administration, including, but not limited to, therapeutic agents (e.g., but not limited to, drugs), diagnostic agents, targeting agents, small organic molecules, oligonucleotides, polypeptides, antibodies, antibody fragments, proteins, carbohydrates such as heparin or hyaluronic acid, or lipids such as glycerolipids, glycolipids, or phospholipids, that can form linkages with active functional groups of the POZ polymers or POZ derivatives of the present disclosure.

[0073] As used herein, "lipid" or "lipid moiety" refers to (i) organic compounds that contain esters of fatty acids or their derivatives and are characterized by being insoluble in water but soluble in many organic solvents, including, but not limited to, simple lipids such as fats, oils and waxes, complex lipids such as phospholipids and glycolipids, cationic lipids, non-cationic lipids, neutral lipids, anionic lipids, and derived lipids such as steroids, and (ii) organic compounds that do not contain esters of fatty acids but that mimic such organic compounds by virtue of their amphiphilic nature, i.e., they possess both hydrophobic and hydrophilic portions and thus can aggregate in specific ways in aqueous environments to form layers, vesicles and LNPs.

[0074] As used herein, "small interfering RNA (siRNA)" refers to a class of double-stranded RNA molecules, 16-40 nucleotides in length, that are involved in the RNA interference (RNAi) pathway to interfere with the expression of specific genes. In addition to their role in the RNAi pathway, siRNAs also function in RNAi-related pathways.

[0075] As used herein, "RNA" refers to a molecule that contains at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, and polyvalent RNA. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides.

[0076] POZ-lipid conjugates The POZ-lipid conjugates of the present disclosure comprise a lipid moiety linked to a polyoxazoline polymer. The components of the POZ-lipid conjugates are described in further detail below.

[0077] POZ part A variety of POZ polymers may be used in the POZ-lipid conjugates of the present disclosure, which are described in more detail below. Generally, however, a POZ polymer may contain a single type or class of functional group, or may contain more than one type or class of functional group. A POZ may be a linear POZ polymer, a branched POZ polymer, a pendent POZ polymer, or a multi-arm POZ polymer. Representative POZ polymers are described in U.S. Patent Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated by reference in its entirety for such teachings. The polyoxazoline polymer can be a homopolymer. Similarly, the polyoxazoline polymer can be a random or block copolymer that includes one or more units of a first polyoxazoline polymer separated by one or more units of a second polyoxazoline polymer. Similarly, the POZ can be a poly(methyloxazoline) (PMOZ), which is fairly hydrophilic, or a poly(ethyloxazoline) (PEOZ), which is less hydrophilic.

[0078] In one embodiment, POZ polymer is prepared by living cationic polymerization.POZ polymer can also be prepared by other methods known in the art.As described in more detail below, POZ can be directly conjugated to lipid, or can be linked to lipid via linker moiety.Any suitable linker moiety can be used for coupling POZ to lipid, including but not limited to ester-free linker moiety and ester-containing linker moiety.In addition, POZ polymer can be conjugated to lipid moiety via suitable chemical group at initiator or terminal end of polymer, or via suitable chemical group at pendant position of polymer, as described in more detail below.

[0079] In the present disclosure, whenever a polyoxazoline derivative or polyoxazoline polymer is described, the polyoxazoline polymer can be characterized by low polydispersity (PD) values ​​and / or increased purity, such that the polymer is useful in pharmaceutical applications. In certain embodiments, the methods of the present disclosure provide polyoxazoline derivatives having low PD values ​​at increased molecular weight (MW) values. For example, in one embodiment, the POZ portion has a molecular weight of about 500 to about 10,000 daltons. In another embodiment, the POZ portion has a molecular weight of about 500 to about 5,000 daltons. In yet another embodiment, the POZ portion has a molecular weight of about 1,000 daltons to about 2,500 daltons. In yet another embodiment, the POZ portion has a molecular weight of about 2,000 daltons to about 5,000 daltons. In yet another embodiment, the POZ portion has a molecular weight of about 5,000 daltons to about 10,000 daltons. In such embodiments, at least one polyoxazoline polymer chain has a polydispersity value of 1.2 or less, 1.1 or less, or 1.05 or less. Methods for synthesizing polyoxazoline polymers and their derivatives having low PD values ​​are described in International Application Nos. PCT / US2008 / 002626 and PCT / US2008 / 078159, which are incorporated by reference in their entireties for such teachings.

[0080] lipid part In one embodiment, the lipid moiety of the POZ-lipid conjugate comprises at least one hydrophobic moiety. In another embodiment, the lipid moiety comprises two hydrophobic moieties. In this aspect, the hydrophobic moiety can be an acyl chain, an alkyl chain, or a combination thereof. The acyl and alkyl chains can vary in length. Furthermore, the acyl and alkyl chains can be saturated or can include one or more unsaturated regions (such as one or more double bonds).

[0081] Regardless of the number of hydrophobic moieties, lipid moiety also comprises a chemical group that can form linkage with the chemical group of POZ polymer.In this aspect, the chemical group can be an amine group, a hydroxyl group, an aldehyde group, a carboxylic acid group, and combinations thereof, but does not exclude other chemical groups.In one embodiment, lipid moiety can comprise a reactive amino group that can be used to form linkage with POZ polymer.

[0082] In some embodiments, the chemical group of the lipid may be located at the hydrophilic head group position, and as described above, the POZ polymer can be conjugated to the lipid via a suitable chemical group at the initiator or terminal end of the polymer, or via a suitable chemical group at a pendant position of the polymer.

[0083] As described in more detail below, the nature of the linkage depends on the chemical groups present on the POZ polymer and the chemical groups present on the lipid moiety. In some embodiments, the linkage is degradable in the presence of certain enzymes. In other embodiments, the linkage is stable in the presence of the same enzymes.

[0084] In one embodiment, the lipid moiety of the POZ-lipid conjugate is an uncharged lipid. For example, any uncharged lipid that can form a layer, vesicle and / or LNP composition alone or in combination with other lipid components is suitable for use in forming the POZ-lipid conjugate of the present disclosure. In another embodiment, the lipid moiety can be synthetic or natural.

[0085] The lipid moiety of the POZ-lipid conjugate can be selected to impart desired characteristics to the LNPs described herein. For example, the degree of unsaturation of the lipid can be selected to provide desired properties to the LNPs described herein. For example, increasing the degree of unsaturation of the lipid moiety can impart fluidity to the LNP composition. Additionally, a cis configuration around the unsaturated region can also impart increased fluidity to the LNP composition. Similarly, a saturated lipid moiety can impart rigidity to the LNP composition. Fluidity and / or rigidity can be selected to at least partially control the stability of the LNP composition and / or the release rate of the POZ-lipid conjugate from the LNP composition.

[0086] In one embodiment, the lipid moiety of the POZ-lipid conjugate is a phospholipid.For example, the lipid moiety of the POZ-lipid conjugate can be phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), or combinations thereof.

[0087] In another embodiment, the lipid moiety of the POZ-lipid conjugate is a glycerolipid. For example, the lipid moiety can be αβ-diacylglycerol.

[0088] In another embodiment, the lipid moiety of the POZ-lipid conjugate is a dialkylamine. For example, the lipid moiety can be dimyristylamine.

[0089] In some embodiments, at least one of the two acyl or alkyl chains of the lipid moiety in the POZ-lipid conjugate is saturated. In some embodiments, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each saturated. In yet another embodiment, one of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is saturated, and the other acyl or alkyl chain is unsaturated. In yet another embodiment, the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate are each unsaturated. When one or more acyl or alkyl chains of the lipid in the POZ-lipid conjugate are unsaturated, the acyl or alkyl chain can contain 1-6, 1-4, 1-3, or 1-2 unsaturated regions. If a double bond is present, it can be in a cis or trans configuration, or a mixture of cis and trans configurations.

[0090] In one embodiment, at least one of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 1-5 carbons in length, 6-10 carbons in length, 11-16 carbons in length, or 17-21 carbons in length. In another embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 1-5 carbons in length, 6-10 carbons in length, 11-16 carbons in length, or 17-21 carbons in length. Such acyl or alkyl chains, regardless of length, include both even and odd chain lengths and may be saturated or unsaturated as described above.

[0091] In one particular embodiment, at least one of the two acyl or alkyl chains of the lipid moiety in the POZ-lipid conjugate is 6 to 10 carbons in length. In another embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 6 to 10 carbons in length. Such acyl or alkyl chains, regardless of length, include both even and odd chain lengths and may be saturated or unsaturated, as described above.

[0092] In another embodiment, at least one of the two acyl or alkyl chains of the lipid moiety in the POZ-lipid conjugate is 11-16 carbons in length. In another embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 11-16 carbons in length. Such acyl or alkyl chains, regardless of length, include both even and odd chain lengths and may be saturated or unsaturated, as described above.

[0093] In yet another embodiment, at least one of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 17-21 carbons in length. In yet another embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 17-21 carbons in length. Such acyl or alkyl chains, regardless of length, include both even and odd chain lengths and may be saturated or unsaturated, as described above.

[0094] In one embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbons in length, and these acyl or alkyl chains are saturated or unsaturated. In another embodiment, each of the two alkyl or acyl chains of the lipid in the POZ-lipid conjugate is 11, 12, 13, 14, 15, or 16 carbons in length, and these acyl chains are saturated or unsaturated. In a further embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is 12, 13, 14, or 15 carbons in length, and these acyl chains are saturated or unsaturated. In yet another embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is an acyl chain of 13 or 14 carbons in length, and the acyl chains are saturated or unsaturated. In yet another embodiment, each of the two alkyl or acyl chains of the lipid in the POZ-lipid conjugate is an alkyl chain of 11, 12, 13, 14, 15, or 16 carbons in length, and the alkyl chains are saturated or unsaturated. In a further embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is an alkyl chain of 12, 13, 14, or 15 carbons in length, and the alkyl chains are saturated or unsaturated. In a further embodiment, each of the two acyl or alkyl chains of the lipid in the POZ-lipid conjugate is an alkyl chain of 13 or 14 carbons in length, and the alkyl chains are saturated or unsaturated.

[0095] In yet another embodiment, each acyl or alkyl chain of the lipid moiety in the POZ-lipid conjugate has the same length and is unsaturated.For example, the acyl or alkyl chain can be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbons long, 11, 12, 13, 14, 15, or 16 carbons long, 12, 13, 14, or 15 carbons long, or 13 or 14 carbons long and unsaturated.

[0096] In another embodiment, the lipid moiety in the POZ-lipid conjugate is 1,2-dimyristoyl-sn-glycerol or 1,2-dilauroyl-sn-glycerol. In yet another embodiment, the lipid moiety in the POZ-lipid conjugate is di(tetradecyl)acetamide or di(dodecyl)acetamide. In yet another embodiment, the lipid moiety in the POZ-lipid conjugate is N,N-di(tetradecyl)acetamide or N,N-di(dodecyl)acetamide. In yet another embodiment, the lipid moiety can be 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE).

[0097] POZ-lipid conjugates Generally, covalent attachment of POZ to lipids is accomplished by reaction of an activated chemical group of the POZ polymer with a complementary chemical group of the lipid. The chemical groups of the POZ polymer and / or lipid may be activated prior to reaction (such as, but not limited to, removal of protecting groups, if present). Hydroxyl, amine, or carboxyl groups may be activated for coupling with monofunctional activating agents, such as N-hydroxysuccinimide, ethyl chloroformate, DCCD, Woodward's reagent K, cyanuric acid, trifluoromethanesulfonyl chloride, among others. Several bifunctional crosslinking reagents, including groups with various reactivities, such as several diisocyanates, may also be used.

[0098] Specific lipids for inclusion in POZ-lipid conjugates are described above and herein.

[0099] In one embodiment, the POZ-lipid conjugates of the present disclosure, or pharma- ceutically acceptable forms thereof, including but not limited to, pharma- ceutically acceptable salts, have the general formula I: R-POZ n -L-lipid I where: R is an initiating group, POZ is a polyoxazoline polymer; n is 1 to 1,000 and represents the number of monomer units comprising the polyoxazoline polymer; L is a linking group that optionally includes a cleavable moiety with controlled cleavage rate, which represents a direct linkage through a reactive group on the lipid and a reactive group on the polymer, where the direct linkage can form a cleavable moiety whose cleavage rate can be controlled from highly unstable to stable; Lipid refers to a lipid moiety as described herein.

[0100] In one embodiment of structure I, the POZ polymer comprises at least one reactive group capable of forming a linkage with a lipid or a linking group. The linkage between the polymer and the lipid (whether a direct linkage or a linkage utilizing a linking group) can be formed between any reactive group on the polymer backbone and a reactive group on the lipid, including reactive groups at terminal or pendent (at terminal) positions. In one aspect, the linkage between the linking group and the polymer can be formed at the terminal end of the polymer. In another aspect, the linkage between the linking group and the polymer can be formed at pendent positions of the polymer. Furthermore, the linkage (whether a direct linkage or a linkage utilizing a linking group) can comprise a component of a reactive group originally present on the polymer or lipid. The linkage (whether a direct linkage or a linkage utilizing a linking group) can be physiologically degradable. In this aspect, the linkage can comprise a cleavable moiety. Suitable linking groups include, but are not limited to, ethers, esters, amines, amides, and combinations thereof.

[0101] In one aspect of this embodiment, L is a stable linkage. In another aspect of this embodiment, L is physiologically degradable and includes a cleavable moiety. For example, in one embodiment, L can be selected from esters, carboxylates (-C(O)-O-), carbonates (-OC(O)-O-), carbamates (-OC(O)-NH-), and amides (-C(O)-NH-). In yet another aspect of this embodiment, L is a linkage that does not include a cleavable moiety.

[0102] Exemplary R groups include, but are not limited to, hydrogen, alkyl, and substituted alkyl. In one embodiment, the initiating group is an alkyl group, such as a C1-C4 alkyl group. In the specific embodiment above, the initiating group is a methyl group. In another embodiment, the initiating group is H. In some embodiments, the initiating group may be selected to lack an active functional group. In other embodiments, the initiating group may be selected to include an active functional group. Additional suitable initiating groups are disclosed in U.S. Patent Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated by reference in its entirety for such teachings.

[0103] In some embodiments, R is H or CH.

[0104] In one embodiment, the POZ polymer in structure I is [N(COR2)CH2CH2] n where R2 is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, and heterocyclylalkyl groups for each repeat unit of the POZ polymer, R is H or CH3, and the degree of polymerization "n" can be in the range of 15 to 35, 20 to 30, or 25.

[0105] In another embodiment, the POZ polymer in structure I is [N(COR2)CH2CH2] n where R2 is independently selected from unsubstituted and substituted alkyl for each repeat unit of the POZ polymer, R is H or CH3, and n can be in the range of 15 to 35, 20 to 30, or 25.

[0106] In yet another embodiment, the POZ polymer in structure I is [N(COR2)CH2CH2] nwhere R2 is independently selected from -CH3 and -CH2-CH3 for each repeat unit of the POZ polymer, and optionally R is H or CH3, and n can be in the range of 15 to 35, 20 to 30, or 25.

[0107] POZ polymer is [N(COR2)CH2CH2] n When the polymer is represented by the formula: POZ polymer of the conjugate is soluble in aqueous environment. The nature of the pendent group (R2) can change the solubility to some extent. For example, when R2 is methyl (like PMOZ), the polymer is highly water-soluble, and when R2 is ethyl (like PEOZ), the polymer remains water-soluble, but to a lesser extent than PMOZ. The solubility of the POZ polymer allows it to extend beyond the liposome surface into the extraliposomal environment. In such a way, the POZ polymer can effectively shield the liposome surface.

[0108] Specific embodiments of structure I above include, but are not limited to, L being an amidase-cleavable amide, shown below as I(a)(1) and I(a)(2).

[0109] [ka]

[0110] In an alternative embodiment of structure I, the same lipid group can be incorporated as a stable amine (rather than an amide) as shown below in I(b).

[0111] [ka]

[0112] In yet another embodiment of structure I, similar lipids can be coupled via relatively labile ester linkages, shown below as I(c) and I(d).

[0113] [ka]

[0114] In yet another embodiment of structure I, lipids may be coupled via relatively labile ether linkages, shown below as I(e) and I(f).

[0115] [ka]

[0116] In one embodiment, for structures I(a) through I(f) (where applicable), m is 1 or 2, n is in the range of 1 to 1000, o is in the range of 1 to 5, and p is in the range of 1 to 10.

[0117] As demonstrated in the Examples section below, the above embodiments of structure I (I(a)-I(f)) hydrolyze at different rates in plasma, thus illustrating one of the key elements of the novel POZ-lipid conjugates of the present disclosure.

[0118] Other specific embodiments of structure I above include, but are not limited to, those depicted below as I(g) and I(h).

[0119] [ka]

[0120] In one embodiment, for structures I(g) through I(h), m is 1 or 2 and n is in the range of 1-1000.

[0121] In another embodiment of the present invention, the POZ-lipid conjugates of the present disclosure, or pharma- ceutically acceptable forms thereof, including but not limited to, pharma- ceutically acceptable salts, have the general formula II: in which the lipid is attached to the polymer chain at the initiator end rather than the terminal end as in formula I: Lipid-L1-(POZ) n a -T II where: POZ is a polyoxazoline polymer of the structure [N(COR2)CH2CH2]; L1 is a linking group that optionally includes a cleavable moiety whose cleavage rate can be controlled, or represents a direct linkage through a reactive group of the lipid and a reactive group of the polymer, the direct linkage being capable of forming a cleavable moiety whose cleavage rate can be controlled; R2 is independently selected for each repeat unit of the polyoxazoline polymer from unsubstituted or substituted alkyl, alkenyl, alkyne-substituted alkyl, aralkyl, heterocyclylalkyl, or an active functional group; T is a terminating end group; a is ran representing a random copolymer or block representing a block copolymer; n is an integer from 1 to 1,000.

[0122] The nature of the pendent group (R2) can change the solubility to some extent. The solubility of the POZ polymer allows the POZ polymer to extend beyond the liposome surface into the extraliposomal environment. In such a manner, the POZ polymer can effectively shield the liposome surface and prevent aggregation during LNP formation. Furthermore, without being bound to any particular theory, it is believed that the POZ-lipid conjugate must be "shed" from the LNP surface after administration to efficiently deliver the nucleic acid payload. In some embodiments, the addition of a highly hydrophilic group to the POZ-lipid conjugate at the R2 position allows additional shielding and / or enhanced shedding of the POZ-lipid conjugate from the LNP during LNP formation and / or administration, facilitating the delivery of the payload. In one embodiment, R2 comprises at least one hydrophilic group. In another embodiment, R2 comprises multiple hydrophilic groups.

[0123] L1 (whether a direct linkage or a linking group) can include a component of a reactive group originally present on the polymer or lipid. Suitable linking groups are described herein. L1 can optionally include a cleavable moiety, such as, but not limited to, an ester, carboxylate (-C(O)-O-), carbonate (-OC(O)-O-), carbamate (-OC(O)-NH-), or amide (-C(O)-NH-).

[0124] Exemplary active functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). In some aspects, the active functional group is a hydrophilic group. In some embodiments, the active functional group is used to add a hydrophilic group to the POZ-lipid conjugate, such as by click chemistry using an alkyne or azide active functional group.

[0125] In one aspect of this embodiment, L1 is a stable linkage. In another aspect of this embodiment, L1 is physiologically degradable and contains a cleavable moiety. In an alternative aspect of this embodiment, L1 is a linkage that does not contain a cleavable moiety. Suitable linkages L1 are described herein.

[0126] In one aspect of this embodiment, L1 is a triazole linking group, as further detailed below.

[0127] In another aspect of this embodiment, T is a terminating nucleophile. For example, T can be ZBQ, where Z is S, O, or N, B is an optional linking group, and Q is a terminating nucleophile or a terminal portion of a nucleophile.

[0128] The B group can include, but is not limited to, an alkylene group. In certain embodiments, B is -(CH2) y -, where y is an integer selected from 1 to 16.

[0129] In certain embodiments, Z is S. The POZ-lipid conjugates containing sulfur groups described herein can be prepared by terminating the POZ cation with a mercaptide reagent, such as, but not limited to, a mercapto-ester (e.g., -S-CH2CH2-CO2CH3) or a mercapto-protected amine (e.g., -S-CH2CH2-NH-tBoc). Such POZ conjugates allow for efficient large-scale ion exchange chromatography purification (to remove secondary amines), as well as control of polydispersity (polydispersity of 1.10 or less) and the creation of conjugates with higher molecular weight POZ polymers. In another embodiment, Z is N. In another embodiment, Z is O.

[0130] In some embodiments, Q is inert (i.e., does not contain a functional group). When Q is an inert group, any inert group can be used, including, but not limited to, -CH, alkyl, and aryl mercaptide groups. In alternative embodiments, Q is or contains an active functional group. When Q is or contains an active functional group, suitable functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). When Q is or contains an active functional group, Q can be the same as R or different from R (i.e., Q and R can be chemically orthogonal to each other).

[0131] In one particular aspect of this embodiment, R2 is independently selected from unsubstituted or substituted alkyl for each repeat unit of the polyoxazoline polymer, optionally R is H or CH3 and n is 15 to 35, 20 to 30, 22 to 28, or 25. In another aspect of this embodiment, R2 is independently selected from CH3 and CH2-CH3 for each repeat unit of the polyoxazoline polymer, optionally R is H or CH3 and n is 15 to 35, 20 to 30, 22 to 28, or 25. In any of the above aspects, T is ZBQ, where Z is S and B is -(CH2). y and Q is an inert group, such as, but not limited to, -CH or an alkyl or aryl mercaptide group. Alternatively, in any of the above embodiments, T is ZBQ, where Z is S and B is -(CH). y - and Q is or includes a functional group, such as, but not limited to, an alkyne, alkene, amine, oxyamine, aldehyde, ketone, acetal, thiol, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active ester), activated carbonate, chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS).

[0132] Specific embodiments of structure II above include, but are not limited to, structure II(a) below, in which a lipid is attached at the initiator end and the terminating nucleophile is sulfur.

[0133] [ka]

[0134] In an alternative embodiment of structure II, shown below as II(b), a lipid is attached to the initiator end and the terminating nucleophile is -OH.

[0135] [ka]

[0136] In an alternative embodiment of structure II, shown below as II(c), a lipid is attached to the initiator end, the terminating nucleophile is nitrogen, and the lipid is attached via a 2-propionate ester.

[0137] [ka]

[0138] Compounds II(a)-II(c) are prepared by the "click" reaction of an azide to a pendent alkyne group as shown below.

[0139] [ka]

[0140] In yet another alternative embodiment of structure II, shown below as II(d), a lipid is attached to the initiator end, the terminating nucleophile is sulfur, and the lipid is attached by an acetate ester.

[0141] [ka]

[0142] II(d) is prepared by the "click" reaction of an azide to an alkyne starting group as shown below.

[0143] [ka]

[0144] In one embodiment, for structures II(a) through II(d) (where applicable), m is 1-2, n is in the range of 1-1000, and o is in the range of 1-5.

[0145] In another embodiment, the POZ-lipid conjugates of the present disclosure, or a pharma- ceutically acceptable form thereof, including but not limited to a pharma- ceutically acceptable salt, may be represented by general formula III: R-(POZ) n a -Z-L2-lipid III During the ceremony, POZ is a polyoxazoline polymer of the structure [N(COR2)CH2CH2]; L2 is a linking group that optionally includes a cleavable moiety whose cleavage rate can be controlled, or represents a direct linkage through a reactive group of the lipid and a reactive group of the polymer, the direct linkage being capable of forming a cleavable moiety whose cleavage rate can be controlled; R is an initiating group, R2 is independently selected for each repeat unit of the polyoxazoline polymer from an unsubstituted or substituted alkyl, alkenyl, alkyne-substituted alkyl, aralkyl, heterocyclylalkyl group, or an active functional group; Z is S, O, or N; a is ran representing a random copolymer or block representing a block copolymer; n is an integer from 1 to 1,000.

[0146] Similar to structure II, the nature of the pendent group (R2) can change the solubility to some extent. The solubility of polyoxazoline polymer allows polyoxazoline polymer to extend beyond the liposome surface into the extraliposomal environment. In such a manner, polyoxazoline polymer can effectively shield the liposome surface. Furthermore, POZ-lipid conjugates must be "shed" from the LNP surface after administration to efficiently deliver nucleic acid payload. In some embodiments, the addition of a highly hydrophilic group to the POZ-lipid conjugate at the R2 position allows additional shielding and / or enhanced shedding of POZ-lipid conjugates from LNPs upon LNP formation and / or administration, facilitating the delivery of payload.

[0147] L2 (whether a direct linkage or a linking group) can include a component of a reactive group originally present on the polymer or lipid. Suitable linking groups are described herein. L2 can optionally include a cleavable moiety, such as, but not limited to, an ester, a carboxylate (-C(O)-O-), a carbonate (-OC(O)-O-), a carbamate (-OC(O)-NH-), or an amide (-C(O)-NH-).

[0148] R groups include, but are not limited to, hydrogen, alkyl, and substituted alkyl. In one embodiment, the initiating group is an alkyl group, such as a C1-C4 alkyl group. In the specific embodiment above, the initiating group is a methyl group. In another embodiment, the initiating group is H. The initiating group may be selected to lack an active functional group. Alternatively, the initiating group may be selected to include an active functional group. Additional exemplary initiating groups are disclosed in U.S. Pat. Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated by reference in its entirety for such teachings.

[0149] Active functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). In some aspects, the active functional group is a hydrophilic group. In some embodiments, the active functional group is used to add a hydrophilic group to the POZ-lipid conjugate, for example, by click chemistry using an alkyne or azide active functional group.

[0150] In one aspect of this embodiment, L2 is a stable linkage. In another aspect of this embodiment, L2 is physiologically degradable and contains a cleavable moiety. In an alternative aspect of this embodiment, L2 is a linkage that does not contain a cleavable moiety. Suitable linkages L2 are described herein.

[0151] In certain embodiments, Z is S. POZ conjugates containing sulfur groups as described herein can be prepared by terminating the POZ cation with a mercaptide reagent, such as, but not limited to, a mercapto-ester (e.g., -S-CH2CH2-CO2CH3) or a mercapto-protected amine (e.g., -S-CH2CH2-NH-tBoc). Such POZ conjugates allow for efficient large-scale ion exchange chromatographic purification (to remove secondary amines), as well as control of polydispersity (polydispersity of 1.10 or less) and the creation of conjugates with higher molecular weight POZ polymers. In another embodiment, Z is N. In another embodiment, Z is O.

[0152] In an aspect of this embodiment, R2 is independently selected from unsubstituted or substituted alkyl for each repeat unit of the polyoxazoline polymer; optionally, R is H or CH3 and n is 15-35, 20-30, or 25.

[0153] In another aspect of this embodiment, R2 is independently selected from CH3 and CH2-CH3 for each repeat unit of the polyoxazoline polymer; optionally, R is H or CH3 and n is 15-35, 20-30, or 25.

[0154] In another embodiment, the POZ-lipid conjugates of the present disclosure, or a pharma- ceutically acceptable form thereof, including but not limited to a pharma- ceutically acceptable salt, may be represented by the general formula IV:

[0155] [ka] During the ceremony, R is an initiating group, R2 is independently selected for each repeat unit of the polyoxazoline polymer from an unsubstituted or substituted alkyl, alkenyl, alkyne-substituted alkyl, aralkyl, heterocyclylalkyl group, or an active functional group; L3 is a linking group that optionally includes a cleavable moiety whose cleavage rate can be controlled, or represents a direct linkage through a reactive group of the lipid and a reactive group of the polymer, the direct linkage being capable of forming a cleavable moiety whose cleavage rate can be controlled; Lipids are lipids, T is a terminating end group; a is ran representing a random copolymer or block representing a block copolymer; m is an integer from 0 to 100; n is an integer from 1 to 5.

[0156] In one embodiment, n is 1 and this monomer unit is the initial unit adjacent to R.

[0157] The nature of the pendent group (R2) can change the solubility to some extent. The solubility of polyoxazoline polymer allows polyoxazoline polymer to extend beyond the liposome surface into the extraliposomal environment. In such a way, polyoxazoline polymer can effectively shield the liposome surface. Furthermore, POZ-lipid conjugates must be "shed" from the LNP surface after administration to efficiently deliver nucleic acid payload. In some embodiments, the addition of a highly hydrophilic group to the POZ-lipid conjugate at the R2 position allows additional shielding and / or enhanced shedding of POZ-lipid conjugates from LNPs upon LNP formation and / or administration, facilitating the delivery of payload.

[0158] L3 (whether a direct linkage or a linking group) can include a component of a reactive group originally present on the polymer or lipid. Suitable linking groups are described herein. L3 can optionally include a cleavable moiety, such as, but not limited to, an ester, carboxylate (-C(O)-O-), carbonate (-OC(O)-O-), carbamate (-OC(O)-NH-), or amide (-C(O)-NH-).

[0159] R groups include, but are not limited to, hydrogen, alkyl, and substituted alkyl. In one embodiment, the initiating group is an alkyl group, such as a C1-C4 alkyl group. In the specific embodiment above, the initiating group is a methyl group. In another embodiment, the initiating group is H. The initiating group may be selected to lack an active functional group. Alternatively, the initiating group may be selected to include an active functional group. Additional exemplary initiating groups are disclosed in U.S. Pat. Nos. 7,943,141, 8,088,884, 8,110,651, 8,101,706, 8,883,211, 9,284,411, and U.S. Patent Application Nos. 13 / 003,306, 13 / 549,312, and 13 / 524,994, each of which is incorporated by reference in its entirety for such teachings.

[0160] Active functional groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). In some aspects, the active functional group is a hydrophilic group. In some embodiments, the active functional group is used to add a hydrophilic group to the POZ-lipid conjugate, for example, by click chemistry using an alkyne or azide active functional group.

[0161] In one aspect of this embodiment, L3 is a stable linkage. In another aspect of this embodiment, L3 is physiologically degradable and comprises a cleavable moiety. In an alternative aspect of this embodiment, L3 is a linkage that does not comprise a cleavable moiety. Suitable linkages L3 are described herein.

[0162] In one aspect of this embodiment, T is a terminating nucleophile. In one aspect of this embodiment, T is ZBQ, where Z is S, O, or N, B is an optional linking group, and Q is a terminating nucleophile or a terminal portion of a nucleophile.

[0163] B groups include, but are not limited to, alkylene groups. In certain embodiments, B is -(CH2) y -, where y is an integer selected from 1 to 16.

[0164] In certain embodiments, Z is S. POZ conjugates containing sulfur groups as described herein can be prepared by terminating the POZ cation with a mercaptide reagent, such as, but not limited to, a mercapto-ester (e.g., -S-CH2CH2-CO2CH3) or a mercapto-protected amine (e.g., -S-CH2CH2-NH-tBoc). Such POZ conjugates allow for efficient large-scale ion exchange chromatographic purification (to remove secondary amines), as well as control of polydispersity (polydispersity of 1.10 or less) and the creation of conjugates with higher molecular weight POZ polymers. In another embodiment, Z is N. In another embodiment, Z is O.

[0165] In some embodiments, Q is inert (i.e., does not contain a functional group). When Q is an inert group, any inert group can be used, including, but not limited to, -CH, alkyl, and aryl mercaptide groups. In other embodiments, Q is or contains an active functional group. When Q is or contains an active functional group, exemplary groups include, but are not limited to, alkynes, alkenes, amines, oxyamines, aldehydes, ketones, acetals, thiols, ketals, maleimides, esters, carboxylic acids, activated carboxylic acids (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active esters), activated carbonates, chloroformates, alcohols, azides, vinyl sulfones, or orthopyridyl disulfides (OPSS). When Q is or contains an active functional group, Q can be the same as R2 or different from R2 (i.e., Q and R2 are chemically orthogonal to each other).

[0166] In an aspect of this embodiment, R2 is independently selected from unsubstituted or substituted alkyl for each repeat unit of the polyoxazoline polymer, optionally, R is H or CH3, m is 15-35, 20-30, or 25, and n is 1. In another aspect of this embodiment, R2 is independently selected from CH3 and CH2-CH3 for each repeat unit of the polyoxazoline polymer, optionally, R is H or CH3, m is 15-35, 20-30, or 25, and n is 1. In any of these aspects, T is ZBQ, where Z is S and B is -(CH2). y and Q is an inert group, such as, but not limited to, -CH or an alkyl or aryl mercaptide group. In any of these embodiments, T is ZBQ, where Z is S and B is -(CH). y- and Q is or includes a functional group, such as, but not limited to, an alkyne, alkene, amine, oxyamine, aldehyde, ketone, acetal, thiol, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active ester), activated carbonate, chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS).

[0167] In specific embodiments of general formula IV, the POZ-lipid conjugate of the present disclosure comprises:

[0168] [ka]

[0169] A similar series of compounds is made by coupling an azide to an initial alkyne group as shown below.

[0170] [ka]

[0171] These compounds contain a triazole ring. There is evidence that this triazole ring is a privileged scaffold and can transport ligands attached to it through the 26S protease, the enzyme responsible for cleaving polypeptides in cells. These so-called proteolytic targeting chimeras (PROTACS) are a promising class of drugs that have been shown to bind to one end of a PROTAC and "knock down" the levels of proteins that shuttle through the 26S protease for cleavage. Without being bound to any particular theory, it is contemplated that POZ polymers incorporating a triazole ring can shuttle the POZ polymer through the 26S protease, thus preventing immune presentation of POZ polymers that do not undergo proteolytic cleavage. Of note, hydrolysis of the degradable ester linkages leaves pendent acid groups attached to the polymer upon release of the lipid. The inventors have found that soluble POZ with these remaining pendent acid groups attached via the triazole ring is non-immunogenic despite being taken up by dendritic cells in the subcutaneous compartment. Furthermore, the C to which rotigotine is attached is not immunogenic, despite being taken up by dendritic cells in the subcutaneous compartment. 14Labeling studies of labeled 20kD POZ polymers have shown that the polymer conjugates are taken up via lymphatic vessels draining the subcutaneous injection site. The labeled conjugates eventually appear in the spleen, where they are taken up almost selectively by the red pulp (macrophage compartment). Without being bound to any particular theory, it is contemplated that the POZ-lipid LNPs of the present disclosure may also be taken up selectively by dendritic cells. In this same context, without being bound to any particular theory, it is contemplated that the POZ-lipid LNPs of the present disclosure may also be taken up selectively by macrophages. If so, the oligonucleotide payload in the LNPs may be preferentially expressed in the dendritic cell / macrophage compartment, which may have implications for immune presentation. In one embodiment, it is worth noting that additional triazole-pendent acid may be directly attached as an R group in the above structure. Without being bound to any particular theory, these groups may further contribute to reducing the immunogenicity of the POZ-lipids contained in the LNPs.

[0172] Additional POZ-lipid conjugates are represented below by Formulas V-VII, in which the POZ polymer is linked to the lipid by L, L1, L2, or L3:

[0173] [ka] During the ceremony, Alkyl1 and Alkyl2 are each independently 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbons in length, 11, 12, 13, 14, 15, 16 carbons in length, 12, 13, 14, 15 carbons in length, or 13 or 14 carbons in length, saturated or unsaturated alkyl chains, preferably Alkyl1 and Alkyl2 have the same number of carbon atoms and are each unsaturated; Acyl 1 and Acyl 2 are each independently a saturated or unsaturated acyl chain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbons in length, 11, 12, 13, 14, 15, or 16 carbons in length, 12, 13, 14, or 15 carbons in length, or 13 or 14 carbons in length, preferably Acyl 1 and Acyl 2 have the same number of carbon atoms and are each unsaturated; j is an integer from 1 to 8.

[0174] Cleavable Linking Group As mentioned above, in some of the above-described embodiments, lipids can be linked to POZ polymers via cleavable linkages. In one aspect, a linking group is provided between the POZ polymer and lipids in a form that includes a cleavable moiety. In other words, the linking group includes a physiologically degradable linkage in that it can be cleaved in a specific environment. For example, the linkage can be cleaved in vivo in a subject after administering the POZ-lipid conjugate of the present disclosure or the LNP composition comprising the POZ-lipid conjugate to the subject.

[0175] In one embodiment, the cleavable moiety is cleaved by a chemical reaction. In an aspect of this embodiment, cleavage is achieved by reduction of an easily reduced group, such as, but not limited to, a disulfide. In another embodiment, the cleavable moiety is cleaved by a substance that is naturally present in the subject or that is induced to be present in the subject. In an aspect of this embodiment, such a substance is an enzyme or a polypeptide. Thus, in one embodiment, the cleavable moiety is cleaved by an enzymatic reaction. In yet another embodiment, the cleavable moiety is cleaved by a combination of the above. The linking group can include a portion of a POZ polymer and / or a portion of a lipid, since such portions react to form the linking group, as described below.

[0176] In this aspect, suitable cleavable moieties include, but are not limited to, esters, carboxylates (-C(O)-O-), carbonates (-OC(O)-O-), carbamates (-OC(O)-NH-) and amides (-C(O)-NH-, including amide groups in peptides). Other releasable moieties are described herein. In certain embodiments, the cleavable moiety is an ester. In another particular embodiment, the cleavable moiety is a carbonate or a carboxylate. Furthermore, the linking group can be a natural amino acid, an unnatural amino acid, or a polymer comprising one or more natural and / or unnatural amino acids. The linking group can include some groups derived from a polymer chain and / or a lipid.

[0177] In some embodiments of the POZ-lipid conjugates of Formulas I-IV, L, L1, L2, and L3 are physiologically degradable linkages and each occurrence comprises a cleavable moiety independently selected from ester, carboxylate (-C(O)-O-), carbonate (-OC(O)-O-), carbamate (-OC(O)-NH-), amide (-C(O)-NH-), and combinations thereof.

[0178] In some other embodiments of the POZ-lipid conjugates of Formulas I-IV, each occurrence of L, L1, L2, and L3 is -(CH2) f -(cleavable moiety)-(CH2) g -, where f and g are each an integer independently selected from 0 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, or 1 to 5. In one embodiment, f and g are each an integer independently selected from 1 to 4. In another embodiment, any of L, L1, L2, and L3 can be a disubstituted triazole as described herein.

[0179] In some embodiments of the POZ-lipid conjugates of Formulas I-IV, each occurrence of L, L1, L2, and L3 is -(CH2) f -C(O)-(CH2) g -, -(CH2)f -C(O)-(CH2) h -NHC(O)-(CH2) g -, -(CH2) f -NH(CO)-(CH2) h -C(O)-(CH2) g -, -(CH2) f -NHC(O)-(CH2) g -, -(CH2) f -C(O)NH-(CH2) g -, -(CH2) f -NHOC(O)-(CH2) g -, -(CH2) f -OC(O)NH-(CH2) g -, -(CH2) f -OC(O)ONH-(CH2) g -, -(CH2) f -NHOC(O)O-(CH2) g -, O-(CH2) h , (CH2) h -O or (CH2) h where f, g, and h are each an integer independently selected from 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, or 0 to 5. In one aspect of this particular embodiment, f, g, and h can each be 0 to 4.

[0180] In another embodiment of the POZ-lipid conjugates of formulas I-IV, L, L1, L2, and L3, each occurrence, is independently selected from disubstituted triazoles containing a cleavable moiety in one of the R3 or R4 groups. The cleavable moiety is preferably present in the R4 group. In a specific embodiment, the disubstituted triazole has the following structure:

[0181] [ka] Where: R3 is a linker that connects the triazole moiety to the polymer chain. R3 may be defined in part by the functional group of the polymer chain. In other words, R3 may include a portion of the functional group of the polymer chain. In one embodiment, R3 is -C(O)-R5-, where R5 is absent or a substituted or unsubstituted alkyl of 1 to 10 carbons in length.

[0182] R4 is a linker that connects the triazole moiety to the lipid. R4 may be partially defined by the functional group of the agent. In other words, R4 may include a portion of the functional group of the lipid. In one embodiment, R4 is -R6-R7-R8-, where R6 is substituted or unsubstituted alkyl, substituted or unsubstituted aralkyl, R7 is a group that includes a cleavable moiety or a portion of a cleavable moiety, and R8 is absent or is O, S, CR c , or NR c and R c is H or substituted or unsubstituted alkyl. In some embodiments, R7 and R8 can combine to form a cleavable moiety. In one embodiment, R7 is -R a -(O)-R b -, -R a -OC(O)-R b -, -R a -C(O)-NH-cyclic-OC(O)-R b - (wherein cyclic represents a substituted or unsubstituted aryl, heterocycloalkyl, heterocycle, or cycloalkyl), -R a -C(O)-NH-(CH)-OC(O)-R b -, -R a -C(O)-R b -, -R a -C(O)-OR b -, -R a -OC(O)-OR b -, -R a -OC(O)-NR 15 -R b - (where R 15 is H or a substituted or unsubstituted C1-C5 alkyl, -R a-CH(OH)-OR b -, -R a -SSR b -, -R a -OP(O)(OR 11 )-OR b - (where R 11 is H or a substituted or unsubstituted C1-C5 alkyl, or -R a -C(O)-NR 15 -R b - (where R 15 is H or a substituted or unsubstituted C1-C5 alkyl; R a and R b are each independently absent or substituted or unsubstituted alkyl. a and R b are each independently absent or a C2-C16 substituted or unsubstituted alkyl. In one embodiment of the above, R6 is a linear substituted or unsubstituted C1-C8 alkyl or a branched substituted or unsubstituted C1-C8 alkyl, and R7 is -R a -C(O)-OR b In another embodiment of the above, R6 is a straight chain substituted or unsubstituted C1-C4 alkyl or a branched substituted or unsubstituted C1-C4 alkyl, and R7 is -R a -C(O)-OR b In one embodiment of the above, R6 is -CH2-, -CH2-CH2-, or -CH2(CH3)-, R7 is -C(O)-O-, and R8 is absent.

[0183] In certain embodiments, R3 is -C(O)-(CH2)3 and R4 is -CH2-C(O)-O-, -CH2-CH2-C(O)-O- or -CH2(CH3)-C(O)-O-.

[0184] In certain embodiments, R3 is -C(O)-(CH2)3 and R4 is -CH2-CH2-OC(O), -CH2-CH2-CH2-OC(O), -CH2-CH2-CO-NH-(C6H4)-OC(O)-.

[0185] In some embodiments of the POZ-lipid conjugates of formulas I-IV, L, L1, L2, and L3, each occurrence, are independently selected from one or more of the cleavable moieties described above.In certain embodiments, when more than one linkage L, L1, L2, or L3 is present in the POZ-lipid conjugate, L, L1, L2, and L3 are each the same.In another particular embodiment, when more than one linkage L, L1, L2, or L3 is present in the POZ-lipid conjugate, at least one L, L1, L2, and L3 is different from the remaining linkages.

[0186] Examples 37 and 38 below reveal that some POZ-lipids made according to the present disclosure degrade when exposed to plasma enzymes and amidases.In particular, the ester linkage that connects POZ to lipids can be controlled to slow down or speed up cleavage to release lipids from POZ-lipids in plasma.Some other POZ-lipids, especially those with amide linkages, do not appear to degrade in plasma, but degrade when exposed to amidases present in certain tissues.Other POZ-lipids, such as those with amine linkages, appear to be stable to both plasma and amidases.

[0187] Thus, the present disclosure includes POZ-lipids that are stable in plasma (e.g., amines, amides), POZ-lipids that can be tailored or controlled to degrade at a desired rate in plasma (e.g., esters), and POZ-lipids that are stable in plasma but degrade in specific environments. In fact, substantially precise control of the rate of degradation can be achieved by carefully selecting the linkage between POZ and lipid. In this aspect, the POZ-lipids of the present disclosure can have controllable degradability in physiological media. For example, in one embodiment, the controllable degradability of the linking group results in a POZ-lipid that is stable in physiological media. In this aspect, the hydrolysis rate of the POZ-lipid, i.e., the time it takes to degrade the POZ-lipid (usually measured by its half-life), is an indicator of the stability / degradability of the linkage between POZ and lipid. In one aspect, the linking group between the POZ polymer and lipid results in a POZ-lipid with a half-life of hydrolysis in 50% human plasma of at least about 120 hours.

[0188] In another embodiment, the controllable degradability of the linking group results in a POZ-lipid that degrades over time in physiological media. In some embodiments, the linking group between the POZ polymer and the lipid results in a POZ-lipid with a hydrolysis half-life of about 10 minutes or less in 50% human plasma. For example, the linking group between the POZ polymer and the lipid can be selected such that the POZ-lipid has a hydrolysis half-life of about 3 minutes to about 7 minutes in 50% human plasma. In other embodiments, the linking group between the POZ polymer and the lipid can be selected such that the POZ-lipid has a hydrolysis half-life of more than about 10 minutes in 50% human plasma. For example, the linking group between the POZ polymer and the lipid can be selected such that the POZ-lipid has a hydrolysis half-life of about 11 minutes to about 8 hours in 50% human plasma. In one embodiment, the POZ-lipid has a hydrolysis half-life of about 2 hours to about 5 hours in 50% human plasma.

[0189] Compositions containing POZ-lipids Since lipids are amphiphilic, i.e., they contain both hydrophobic and hydrophilic parts, these molecules can aggregate in specific ways in an aqueous environment to form layers, vesicles, and LNPs. For example, phospholipids are a type of lipid that has such amphiphilic properties, i.e., the head group of the phospholipid is hydrophilic and the tail group is hydrophobic. The hydrophilic head group contains a negatively charged phosphate group and can contain other polar groups. The hydrophobic tail group generally contains a long fatty acid hydrocarbon chain. When placed in an aqueous environment, phospholipids form a variety of structures depending on the specific properties of the phospholipid.

[0190] Thus, the POZ-lipids of the present disclosure are particularly well suited for forming LNP compositions. Also, the LNP compositions of the present disclosure incorporating the POZ-lipid conjugates described herein provide several advantages over similar LNP compositions that do not incorporate the POZ-lipid polymers of the present disclosure. For example, the LNP compositions of the present disclosure are substantially non-immunogenic. In one embodiment, the POZ-lipid conjugates of the present disclosure, when incorporated into the LNP compositions described herein, do not elicit a significant immune response, including but not limited to, the generation of IgM and / or IgG antibodies specific to POZ. In another embodiment, the POZ-lipid conjugates of the present disclosure, when incorporated into the LNP compositions described herein, reduce the immune response, including but not limited to, the generation of IgM and / or IgG antibodies specific to the polymer moiety, compared to the corresponding LNP compositions incorporating PEG-lipid conjugates. In another embodiment, the LNP composition comprising a POZ-lipid conjugate of the present disclosure is present in the blood or tissue of the subject at a concentration of at least 75%, e.g., 80%, 85%, 90%, 95%, or more, after the second administration compared to the first administration. In another embodiment, the LNP composition comprising a POZ-lipid conjugate of the present disclosure is not subject to accelerated blood clearance.

[0191] Any of the POZ-lipid conjugates of the present disclosure may be used in preparing LNP compositions according to the present disclosure. In one embodiment, the LNP composition may be formed with at least one of the POZ-lipid conjugates of the present disclosure and a cationic or ionizable lipid. For example, the composition may be formed with the POZ-lipid conjugates of the present disclosure and a cationic lipid. In another embodiment, the composition may be formed with the POZ-lipid conjugates of the present disclosure and an ionizable lipid. In yet another embodiment, the composition according to the present disclosure comprises the POZ-lipid conjugates of the present disclosure, a cationic or ionizable lipid, and other lipid components (non-derivatized lipids) that can form vesicles and / or liposomes (lacking a polyoxazoline component). Examples of suitable non-derivatized lipids include, but are not limited to, helper lipids and lipids that stabilize the composition.

[0192] In one aspect, an LNP formed in accordance with the present disclosure comprises a POZ-lipid conjugate of the present disclosure, a cationic or ionizable lipid, and (i) a helper lipid to provide structural support and promote endocytosis and / or (ii) a sterol lipid for stability.

[0193] The LNPs formed according to the present disclosure can also include a payload. In this aspect, the payload can be an oligonucleotide, a protein, or a combination thereof. For example, in the specific embodiment shown in FIG. 1, the LNPs of the present disclosure can include (i) an ionizable lipid; (ii) a helper lipid; (iii) a sterol lipid; (iii) a POZ-lipid of the present disclosure; and (iv) an oligonucleotide. In another specific embodiment (not shown), the LNPs of the present disclosure can include a cationic lipid, a helper lipid, a sterol lipid, a POZ-lipid of the present disclosure, and an oligonucleotide. In yet another specific embodiment (not shown), the LNPs of the present disclosure can include a cationic or ionizable lipid, a helper lipid, a sterol lipid, a POZ-lipid of the present disclosure, and a protein.

[0194] In one embodiment, the oligonucleotide comprises DNA, siRNA, self-replicating mRNA, mRNA composed of modified nucleosides, and mRNA composed of natural nucleosides. In one embodiment, the oligonucleotide is DNA. In another embodiment, the oligonucleotide is siRNA. In yet another embodiment, the oligonucleotide is self-replicating mRNA, mRNA composed of modified nucleosides, or mRNA composed of natural nucleosides.

[0195] In one embodiment, when incorporated into the LNP composition, the POZ-lipid conjugate is present in a molar ratio of about 0.25 to about 5 mol % in the lipid layer of the LNP composition, a molar ratio of about 0.5 to about 3 mol % in the lipid layer of the LNP composition, a molar ratio of about 0.75 to about 2 mol % in the lipid layer of the LNP composition, or a molar ratio of about 0.8 to about 1.5 mol % in the lipid layer of the LNP composition.

[0196] A non-limiting example of a cationic lipid suitable for use according to the present invention is 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). Suitable ionizable lipids include, but are not limited to, MC3 98, Lipid 319, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Moderna Lipid H, SM-102, Acuitas A9

[59] , Arcturus Lipid 2,2 (8,8) 4C CH3, Genevant CL1. In one embodiment, the cationic or ionizable lipid has a pKa in the range of 6-7 as measured by TNS dye binding assay.

[0197] Helper lipids refer to amphipathic lipids that have hydrophobic and polar head group moieties and can spontaneously form bilayer vesicles in water, as exemplified by phospholipids, or can stably incorporate into lipid bilayers, with the hydrophobic moiety in contact with the inner hydrophobic region of the bilayer membrane and the polar head group moiety oriented toward the outer polar surface of the membrane. Such helper lipids typically contain one or two hydrophobic acyl hydrocarbon chains or steroid groups and may contain chemically reactive groups in the polar head groups, such as amines, acids, esters, aldehydes, or alcohols. Non-limiting examples include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), in which the two hydrocarbon chains are typically about 14-22 carbon atoms long and have variable degrees of unsaturation. Other suitable helper lipids include, but are not limited to, glycolipids, such as cerebrosides and gangliosides. In one embodiment, the helper lipid is at least one of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).

[0198] A suitable sterol lipid for use according to the present disclosure is cholesterol. In a particular embodiment, the LNP according to the present disclosure comprises a cationic or ionizable lipid in combination with (i) DSPC; (ii) cholesterol; (iii) a POZ-lipid of the present disclosure; and (iv) an oligonucleotide.

[0199] It is important to note that modifications or additions (even very minor modifications or additions) to the LNP composition of the present disclosure can affect not only the structure of the LNP, but also the delivery of the encapsulated payload. For example, when a sterol lipid such as cholesterol is included in the LNP composition together with an ionizable lipid and the POZ-lipid of the present disclosure, the resulting LNP has a single bilayer. When a plant sterol is added, the structure of the LNP becomes more complex and therefore can deliver the payload differently. In this context, the composition of the present disclosure that includes a POZ-lipid conjugate can be monolayer or non-monolayer.

[0200] The particle size of LNPs made according to the present disclosure can vary. In one embodiment, LNPs formed according to the present disclosure are amphiphilic spherical vesicles with a size in the range of about 10 nm to about 10 microns formed by one or more lipid bilayers encasing an aqueous core. In another embodiment, LNPs formed according to the present disclosure have a particle size of about 25 nm to about 8 microns. In yet another embodiment, LNPs formed according to the present disclosure have a particle size of about 30 nm to about 5 microns. In this aspect, the particle size of the LNPs can be about 20 nm to about 3 microns. In another embodiment, the LNPs can be about 10 nm to about 1000 nm, about 25 nm to about 500 nm, about 35 nm to about 250 nm, about 40 nm to about 150 nm, or about 45 nm to about 100 nm. Size fractionation methods are disclosed herein. However, in some aspects, size fractionation is not required.

[0201] The LNP compositions of the present disclosure may be prepared by a variety of methods. In one embodiment, liposomes are prepared by the reverse phase evaporation method (Szoka et al., PNAS 1978, Vol. 75, 4194-4198; Smirnov et al., Byulleten' Experimental'noi Biologii i Meditsiny, 1984, Vol. 98, pp. 249-252; U.S. Patent No. 4,235,871). In this method, an organic solution of liposome-forming lipids, which may include polyoxazoline-lipid conjugates with or without linked targeting molecules, is mixed with a smaller volume of aqueous medium and the mixture is dispersed to form a water-in-oil emulsion, preferably using pyrogen-free ingredients. The targeting molecule to be delivered is added to the lipid solution in the case of lipophilic targeting molecules or to the aqueous medium in the case of water-soluble targeting molecules. The lipid solvent is evaporated off and the resulting gel is converted into liposomes. Reverse phase evaporation vesicles (REVs) have a typical average size of about 0.2-0.4 microns and are predominantly oligolamellar, i.e., contain one or several lipid bilayer shells. REVs can be readily sized by extrusion, as described below, to obtain oligolamellar vesicles having a selected size, preferably about 0.05-0.2 microns.

[0202] Additionally, multilamellar vesicles (MLVs) can be created. In this method, a mixture of liposome-forming lipids, which may include polyoxazoline-lipid conjugates with or without linked targeting molecules as described herein, is dissolved in a suitable solvent and the solvent is evaporated in a vessel to form a thin film. The thin film is then covered with an aqueous medium. The lipid film hydrates to form MLVs. MLVs generally exhibit a size of about 0.1 to 10 microns. MLVs can be sized to the desired size range by extrusion and other methods described herein.

[0203] One sizing method that is effective for REVs and MLVs is to extrude an aqueous suspension of liposomes through a polycarbonate membrane with a selected uniform pore size, typically 0.05, 0.08, 0.1, 0.2, or 0.4 microns (Szoka et al., PNAS 1978, vol. 75, 4194-4198). The pore size of the membrane roughly corresponds to the maximum size of the liposomes produced by extrusion through that membrane, especially if the preparation is one that has been extruded through the same membrane more than once. A method for sizing larger sized MLVs is provided by Zhu et al. (PLoS One. 2009;4(4):e5009. Epub 2009 Apr 6).

[0204] When small particle sizes are desired, REV or MLV preparations can be processed to produce small unilamellar vesicles (SUVs), characterized by sizes in the 0.04-0.08 micron range. Such particles can be useful in targeting tumor or lung tissue if the particles can be absorbed through the capillary walls (particles larger than 0.1 microns may not be absorbed).

[0205] Furthermore, the POZ-lipid conjugates can be introduced into the LNP composition after liposomes are formed using the techniques described above. In this approach, preformed liposomes are incubated in the presence of the POZ-lipid conjugates. The POZ-lipid conjugates are incorporated into the liposomes by diffusion. The concentration of the POZ-lipid conjugates, either free in solution or incorporated by the liposomes, can be monitored, and the method can be terminated when the desired concentration of the POZ-lipid conjugates in the LNP composition is reached. The incubation solution can include surfactants or other agents to facilitate the diffusion of the POZ-lipid conjugates into the LNP composition.

[0206] The LNP composition may be treated to remove extraneous components prior to use. For example, if a detergent is used as described above, excess detergent may be removed prior to use. Additionally, if a payload, such as an oligonucleotide as described above, is entrapped in the LNP composition, excess or unentrapped payload may be removed prior to use. Separation techniques to accomplish this task are known in the art, and the selection of a particular method may depend on the nature of the components to be removed. Suitable methods include, but are not limited to, centrifugation, dialysis, and molecular sieve chromatography. The composition may be sterilized by filtration through a conventional 0.22 micron depth filter.

[0207] LNPs can be prepared by traditional methods, which involve hydrating a lipid film containing POZ-lipid conjugates, ionizable lipids, helper lipids, and cholesterol. This method involves dissolving these materials in an organic solvent, such as chloroform or dichloromethane, and then evaporating the solvent to produce a thin film. The thin film is then hydrated with a buffered aqueous solution containing drugs or nucleic acids to passively encapsulate the payload. LNPs are usually formed as heterogeneous particles with low encapsulation, and require size reduction by extrusion or sonication.

[0208] Another suitable technique uses rapid mixing with a microfluidizer. A lipid stock solution is prepared by dissolving lipids in an organic solvent such as ethanol. The stock aqueous solution contains nucleic acid dissolved in a buffer with known pH, ionic strength and buffering capacity. The two stock solutions are passed through a micromixer at a predetermined speed to allow the cationic lipids to interact with the negatively charged nucleic acid, resulting in higher encapsulation efficiency (i.e., >90%) and uniform size distribution. The ratio of aqueous solvent to organic solvent during the mixing process is important. The organic solvent is removed by dialysis, tangential flow filtration or centrifugation, or other techniques. LNPs of defined size are produced by controlling the microfluidic operation parameters, resulting in LNPs with low polydispersity and uniform particle size. The mean particle diameter (<100 nm), polydispersity (<0.40 and more specifically <0.20), and zeta potential of LNPs are three methods used to characterize the preparations.

[0209] The ratio of POZ-lipid to ionic lipid to cholesterol can be varied to allow for optimal size, high payload release and transfection, and improved stability of the hydrated formulation. In one embodiment, the mol% of POZ-lipid in the LNP is about 0.5-60%. In another embodiment, the mol% of POZ-lipid is about 1-40%. In yet another embodiment, the POZ-lipid is present in an amount of less than about 10% of the total amount of lipid in the LNP. In this embodiment, the POZ-lipid can be present in an amount of about 0.5-5%, about 1-4%, or about 1.5-3.5%. In this embodiment, the remainder of the LNP composition can be about 35-50% sterol lipid, about 30-70% cationic lipid, and about 5-15% helper lipid.

[0210] In one embodiment, the LNP composition comprises a lipid bilayer comprising at least one POZ-lipid conjugate encapsulating an aqueous core comprising oligonucleotides, and the POZ has an average molecular weight of about 0.5-5 kDa. In another embodiment, the LNP composition comprises a lipid bilayer comprising at least one POZ-lipid conjugate encapsulating an aqueous core comprising oligonucleotides, and the POZ has an average molecular weight of about 2-5 kDa.

[0211] Oligonucleotides may be encapsulated in LNPs with high efficiency. In one embodiment, the oligonucleotides are encapsulated in LNPs with at least 90% efficiency. In another embodiment, the oligonucleotides are encapsulated in LNPs with about 90 to about 99% efficiency. In yet another embodiment, the oligonucleotides are encapsulated in LNPs with about 90 to about 95% efficiency. In yet another embodiment, the oligonucleotides are encapsulated in LNPs with greater than about 95% efficiency.

[0212] Administration The LNPs of the present disclosure can be delivered to cells. After administration of the LNPs in vivo, the oligonucleotides are released. In this embodiment, the LNPs of the present disclosure can be included in a pharmaceutical composition that can induce treatment for a disorder or disease. For example, a pharmaceutical composition comprising the LNPs made according to the present disclosure can be used to prevent or treat infectious diseases, including but not limited to SARS-CoV-2, rabies, influenza, and the like. Furthermore, a pharmaceutical composition comprising the LNPs made according to the present disclosure can be used as a therapeutic agent for cancer and genetic diseases. Such pharmaceutical compositions can also include a pharma- ceutically acceptable carrier in addition to the LNPs.

[0213] In one embodiment, a pharmaceutical composition comprising an effective amount of the LNPs of the present disclosure can be delivered to an animal. In this aspect, delivery of the effective amount of the LNPs of the present disclosure can be via subcutaneous, intravenous, intramuscular, intradermal or aerosol routes. In one embodiment, the animal is a human. EXAMPLES

[0214] The following non-limiting examples are merely illustrative of preferred embodiments of the invention and should not be construed as limiting the invention, the scope of which is defined in the claims.

[0215] material 1,2-Dimyristoyl-rac-glycerol (DMG) was purchased from Bachem. PEOZ-propargylamide 2K, 3-azidopropionyl chloride, 3-(2-aminoethoxy)propane-1,2-diol were synthesized by Serina Therapeutics, Inc. Solketal (1,2-isopropylidineglycerol), myristic acid, copper(I) iodide, triethylamine (TEA), and anhydrous pyridine were purchased from Sigma-Aldrich. Anhydrous sodium sulfate, anhydrous magnesium sulfate, tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile (ACN), and diethyl ether were purchased from EMD Millipore. Sodium chloride (NaCl) was purchased from Fluka. Ambersep M4195 (or Dowex M4195) was purchased from Supelco. SNAP Ultra 25 g columns and the Isolera System for column purification were purchased from Biotage.

[0216] Example 1. Synthesis of PMOZ-dimyristylamide (2.2 kD compound 15a)

[0217] [ka]

[0218] An oven-dried 250 mL round bottom flask was charged under an argon atmosphere with PMOZ-COOH (12.00 grams, 5.45 mmol, 1.00 equiv), then DCM (50 mL), N-hydroxysuccinimide (1.25 grams, 10.9 mmol, 2.00 equiv), and finally DMAP (0.033 grams, 0.28 mmol, 0.05 equiv). DCC (2.25 g, 10.9 mmol, 2.00 equiv) was added in one portion and the resulting solution was allowed to stir at room temperature for at least 12 hours. Following this time, the reaction mixture was filtered through a coarse sintered glass frit, then the frit was rinsed with additional DCM. The resulting solution was then slowly transferred to a beaker containing a stirred solution of Et2O (2000 mL). The precipitate was collected by vacuum filtration and the solid was dried under vacuum. The solid was then dissolved in DCM (50 mL) in a 250 mL dry round bottom flask equipped with a stir bar under an argon atmosphere. The reaction mixture was then charged with dimyristylamine (4.46 g, 10.9 mmol, 2.00 equiv.) followed by NEt3 (1.52 mL, 10.9 mmol, 2.00 equiv.) and the reaction mixture was allowed to stir at room temperature for at least 12 hours. After this time, the reaction mixture was precipitated into a beaker containing a stirred solution of 2000 mL of diethyl ether. The solid was collected by vacuum filtration and dried under vacuum. The solid was then dissolved in 200 mL of deionized water and passed through an Amberlite column containing 200 grams of Amberlite IR-67 and 200 grams of Amberlite IR-120H. The filtrate was collected until the aqueous solution gave a negative PAA test. The resulting solution was then passed through a 1000 mL DEAE column and eluted with deionized water until a PAA test gave a negative result. The resulting aqueous solution was concentrated to dryness on a rotary evaporator. The residue was dissolved in dichloromethane (100 mL), dried over sodium sulfate, and precipitated into a beaker containing a stirred solution of 2000 mL of Et2O. The solid was collected by vacuum filtration and dried under vacuum to give 10.9 grams of the title compound. 1H NMR analysis showed standard backbone signals for PMOZ (500 MHz, DMSO) δ 7.92 (m, NH-terminal signal); 3.34 (CH2CH2 backbone); 3.21 (N-CH2); 2.75-2.72 (S-CH2); 1.98 (-CH3); 1.80 (CH2-CO2R). Additional signals for the lipid moiety were present at δ 1.49-1.41 (N-(CH2)2); 1.24 (CH2); 0.85 (CH3).

[0219] Example 2. PEOZ(2.2kD)-Dimyristylamide-2.2kD Compound 2b.

[0220] [ka]

[0221] Compound 2B was prepared via the same procedure used to prepare compound 15B. 1.3 grams of material was isolated in this iteration. 1 H NMR. HNMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 3.64 (CH2CH2 backbone); 3.27 (N-CH2); 2.72 (S-CH2); 2.32-2.19 (C(O)-CH2); 1.12 (CH3). Additional signals for lipid moieties were present at δ 1.49-1.54 (N-(CH2)2); 1.27 (CH2); 0.87 (CH3). A signal for pendant groups was present at δ 1.65 ppm (CH2). The number of pendant groups was previously calculated to be 1.21 from the polymer starting material.

[0222] Example 3 - Synthesis of PMOZ-dimyristylamide, compound 16a, 1.3 pendant groups, 2.2 kD.

[0223] [ka]

[0224] This compound was prepared in a manner similar to that described above for PMOZ 2.2 kD dymyristylamide. 7.7 g was isolated. 1 H NMR analysis showed standard backbone signals for PMOZ (500 MHz, DMSO) δ 7.90 (m, NH terminal signal); 3.33 (CH2CH2 backbone); 3.21 (N-CH2); 2.75-2.72 (S-CH2); 1.98 (-CH3); 1.80 (CH2-CO2R). Additional signals for the lipid moiety were present at δ 1.49-1.41 (N-(CH2)2); 1.24 (CH2); 0.85 (CH3). A signal for the pendant group was present at δ 1.65 ppm (CH2). The number of pendant groups was calculated via comparison of the integrals of the polymer backbone and the pendant group signals. In this case, the number of pendant groups was determined to be 1.35.

[0225] Example 4. Synthesis of PEOZ-dimyristylamide, compound 16b, 1.5 pendant groups, 2.2 kD

[0226] [ka]

[0227] This compound was prepared in a manner similar to that described above for the PMOZ 2.2 kD dymyristylamide. 7.0 was isolated. 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 3.64 (CH2CH2 backbone); 3.27 (N-CH2); 2.72 (S-CH2); 2.32-2.19 (C(O)-CH2); 1.12 (CH3). Additional signals for lipid moieties were present at δ 1.49-1.54 (N-(CH2)2); 1.27 (CH2); 0.87 (CH3). A signal for pendant groups was present at δ 1.65 ppm (CH2). The number of pendant groups was calculated via comparison of the integrals of the polymer backbone and the pendant group signals. In this case, the number of pendant groups was determined to be 1.50.

[0228] Example 5. Synthesis of PEOZ-dimyristoyl glycerol ether, compound 17b, 5 kD.

[0229] [ka]

[0230] The NHS ester (2.6 grams, 0.49 mmol, 1.00 equiv., synthesized similarly to U.S. Pat. No. 7,943,141) was dissolved in DCM (30 mL) in a dry 250 mL round bottom flask equipped with a stir bar under an argon atmosphere. The reaction mixture was then charged with a solution of 3-(2-aminoethoxy)propane-1,2-diol (0.200 grams, 1.48 mmol, 3.00 equiv.) and NEt3 (0.68 mL, 4.9 mmol, 10 equiv.) in 3 mL of DMF, and the reaction mixture was allowed to stir at room temperature for at least 12 hours. After this time, the reaction mixture was precipitated into a beaker containing a stirred solution of 2000 mL of diethyl ether. The solid was collected by vacuum filtration and dried under vacuum. 2.3 grams of material was isolated. 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). Additional signals for the bis-hydroxyamide moiety were present at δ 4.59 (OH) and 4.46 (OH).

[0231] [ka]

[0232] The amide described above (2.3 grams, 0.43 mmol, 1.00 equiv.) was charged to a 100 mL round bottom flask under an argon atmosphere. The material was dissolved in 30 mL of DCM. Myristic acid (0.49 grams, 2.123 mmol, 5.00 equiv.) was added, followed by DMAP (0.01 g, 0.09 mmol, 0.2 equiv.), and finally DCC (0.44 grams, 2.13 mmol, 5.00 equiv.) and the reaction mixture was allowed to stir overnight under an argon atmosphere. The next morning, the reaction mixture was filtered and precipitated into a beaker containing 1600 mL of diethyl ether. The solid was collected by vacuum filtration. The product was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (1.2 grams). 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02(S-CH2); 2.32-2.27 (C(O)-CH2); 0.96(CH3). Additional signals for lipid derivative moieties were present at δ 5.10(CH); 4.25(CH2); 4.08(CH2); 1.48(C(O)CH2); 1.22(CH2); 0.84(CH3).

[0233] Example 6. Synthesis of compound 15b

[0234] [ka]

[0235] An oven-dried 250 mL round bottom flask was charged under an argon atmosphere with PEOZ-COOH (7.00 grams, 3.18 mmol, 1.00 equiv), then DCM (50 mL), N-hydroxysuccinimide (0.48 grams, 4.14 mmol, 1.30 equiv), and finally DMAP (0.04 grams, 0.31 mmol, 0.1 equiv). DCC (0.854 g, 4.14 mmol, 1.30 equiv) was added in one portion and the resulting solution was allowed to stir at room temperature for at least 12 hours. Following this time, the reaction mixture was filtered through a coarse sintered glass frit, then the frit was rinsed with additional DCM. The resulting solution was then slowly transferred to a beaker containing a stirred solution of Et2O (2000 mL). The precipitate was collected by vacuum filtration and the solid was dried under vacuum. The solid was then dissolved in DCM (50 mL) in a 250 mL dry round bottom flask equipped with a stir bar under an argon atmosphere. The reaction mixture was then charged with dimyristylamine (2.6 g, 6.36 mmol, 2.00 equiv.) followed by NEt3 (0.89 mL, 6.36 mmol, 2.00 equiv.) and the reaction mixture was allowed to stir at room temperature for at least 12 hours. After this time, the reaction mixture was precipitated into a beaker containing a stirred solution of 2000 mL of hexane. The solid was collected by vacuum filtration and dried under vacuum. The solid was then dissolved in 200 mL of deionized water and passed through an Amberlite column containing 200 grams of Amberlite IR-67 and 200 grams of Amberlite IR-120H. The resulting aqueous solution was concentrated to dryness on a rotary evaporator. The residue was dissolved in dichloromethane (100 mL), dried over sodium sulfate, and concentrated to give 4.5 grams of the title compound. 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 3.64 (CH2CH2 backbone); 3.27 (N-CH2); 2.72 (S-CH2); 2.32-2.19 (C(O)-CH2); 1.12 (CH3). Additional signals for the lipid moiety were present at δ 1.49-1.54(N-(CH2)2); 1.27(CH2); 0.87(CH3).

[0236] Example 7. Synthesis of compound 17a

[0237] [ka]

[0238] PMOZ 5K NHS ester was prepared in a manner similar to that described above for PEOZ 5K NHS. 3.0 grams were isolated. 1 H NMR analysis showed standard backbone signals for PMOZ (500 MHz, DMSO) δ 7.92 (m, NH terminal signal); 3.34 (CH2CH2 backbone); 3.21 (N-CH2); 2.75-2.72 (S-CH2); 1.98 (-CH3); 1.80 (CH2-CO2R). An additional signal for the NHS moiety was present at δ 2.81 (CH2).

[0239] [ka]

[0240] PMOZ 5K amide was prepared in a manner similar to that described above. HNMR analysis revealed the product material to be an inseparable mixture of approximately 50:50 product and starting NHS ester. This mixture was carried on to the next step without further purification. 1H NMR analysis showed standard backbone signals for PMOZ (500 MHz, DMSO) δ 7.92 (m, NH terminal signal); 3.34 (CH2CH2 backbone); 3.21 (N-CH2); 2.75-2.72 (S-CH2); 1.98 (-CH3); 1.80 (CH2-CO2R). Additional signals for the diol moiety were present at δ 4.61 (CH) and 4.48 (CH2).

[0241] [ka]

[0242] PMOZ 5k dimyristoyl glycerol ether was prepared in a similar manner as described above. 740 grams were isolated. 1 H NMR. HNMR analysis showed standard backbone signals for PMOZ (500 MHz, DMSO) δ 7.92 (m, NH-terminal signal); 3.34 (CH2CH2 backbone); 3.21 (N-CH2); 2.75-2.72 (S-CH2); 1.98 (-CH3); 1.80 (CH2-CO2R). Additional signals for the lipid derivative moiety were present at δ 5.10(CH); 4.26(CH2); 4.08(CH2); 1.49(C(O)CH2); 1.23(CH2); 0.84(CH3).

[0243] Example 8. Synthesis of compound 17c

[0244] [ka]

[0245] PEOZ 2K dimyristoyl glycerol ether was prepared in the same manner as described for PEOZ 5K dimyristoyl glycerol ether. 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02(S-CH2); 2.32-2.27 (C(O)-CH2); 0.96(CH3). Additional signals for lipid derivative moieties were present at δ 5.10(CH); 4.25(CH2); 4.08(CH2); 1.48(C(O)CH2); 1.22(CH2); 0.84(CH3).

[0246] Example 9. Synthesis of compound 18a

[0247] [ka]

[0248] The NHS ester (synthesis previously described, 1.4 grams, 0.67 mmol, 1.00 equiv.) was dissolved in DCM (30 mL) in a dry 250 mL round bottom flask equipped with a stir bar under an argon atmosphere. The reaction mixture was then charged with ethanolamine (0.15 mL, 2.00 mmol, 3.00 equiv.), followed by triethylamine (0.28 mL, 2.00 mmol, 3.00 equiv.), and the reaction mixture was allowed to stir at room temperature for at least 12 hours. After this time, the reaction mixture was precipitated into a beaker containing a stirred solution of 1500 mL of diethyl ether. The solid was collected by vacuum filtration and dried under vacuum. 1.1 grams of material was isolated. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02(S-CH2); 2.32-2.27 (C(O)-CH2); 0.96(CH3). Additional signals for ethanolamine amide were present at δ 4.63(OH); 3.08(N-CH2); 2.69(HO-CH2).

[0249] [ka]

[0250] The amide described above (1 gram, 0.45 mmol, 1.00 equiv.) was charged to a 100 mL round bottom flask under an argon atmosphere. The material was dissolved in 30 mL of DCM. Myristic acid (0.52 grams, 2.27 mmol, 5.00 equiv.) was added, followed by DMAP (0.01 g, 0.09 mmol, 0.2 equiv.), and finally DCC (0.47 grams, 2.27 mmol, 5.00 equiv.) and the reaction mixture was allowed to stir overnight under an argon atmosphere. The next morning, the reaction mixture was filtered and precipitated into a beaker containing 1600 mL of diethyl ether. The solid was collected by vacuum filtration. The product was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (0.740 grams). 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 3.45 (CH2CH2 backbone); 2.74 (S-CH2); 2.40-2.30 (C(O)-CH2); 1.12 (CH3). Additional signals for the lipid moiety were present at δ 4.10 (O-CH2); 2.85 (N-CH2); 1.99 (lipid CH2); 1.58 (lipid CH2); 1.24 (lipid CH2); 0.87 (lipid CH3).

[0251] Example 10. Synthesis of compound 19a

[0252] [ka]

[0253] The NHS ester (synthesis previously described, 6.00 grams, 2.86 mmol, 1.00 equiv.) was dissolved in DCM (100 mL) in a dry 250 mL round bottom flask equipped with a stir bar under an argon atmosphere. The reaction mixture was then charged with propargylamine (0.55 mL, 8.57 mmol, 3.00 equiv.), followed by triethylamine (1.19 mL, 8.57 mmol, 3.00 equiv.), and the reaction mixture was allowed to stir at room temperature for at least 12 hours. After this time, the reaction mixture was precipitated into a beaker containing a stirred solution of 2500 mL of diethyl ether. The solid was collected by vacuum filtration and dried under vacuum. 1.8 grams of material was isolated. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02(S-CH2); 2.32-2.27 (C(O)-CH2); 0.96(CH3). Additional signals for propargylamide were present at δ 3.85(N-CH2); 3.75(CCH).

[0254] [ka]

[0255] A 100 mL round bottom flask was charged with the amide described above (0.92 g, 0.44 mmol, 1.00 equiv), then the azidoacetate (0.39 g, 0.66 mmol, 1.50 equiv), THF (50 mL), CuI (0.34 g, 1.76 mmol, 4.00 equiv), and finally triethylamine (0.62 mL, 4.40 mmol, 10.0 equiv). The resulting mixture was heated to 50° C. and stirred overnight. Following this time, the reaction mixture was cooled to room temperature and quenched by the addition of 0.1 M HCl (30 mL) and stirred for 10 min. The resulting mixture was passed through a pre-prepared 60 mL Dowex column. The resulting solution was concentrated on a rotary evaporator at 35° C. and subsequently transferred to a separatory funnel. Brine (20 mL) was added and the mixture was extracted three times with 20 mL portions of DCM. The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The product was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (0.540 grams). 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 7.71 (NH terminus); 3.45 (CH2CH2 backbone); 2.72 (S-CH2); 2.40-2.30 (C(O)-CH2); 1.12 (CH3). Additional signals for the lipid moiety were present at δ 5.16 (CH); 4.43 (CH2); 4.11 (CH2); 2.29 (N-CH2); 1.61 (lipid CH2); 1.25 (lipid CH2); 0.88 (lipid CH3). Additional signals for the triazole moiety were present at δ 5.29 (triazole CH); 4.53 (ester CH2); 4.23 (NH-CH2).

[0256] Example 11. Synthesis of compound 1b

[0257] [ka]

[0258] An oven-dried 500 mL round bottom flask was charged with PhCl (300 mL) under an argon atmosphere, followed by a stir bar and 2-(5-pentynyl)-2-oxazoline (5.40 mL, 39.79 mmol, 1.20 equiv). TfOH (2.94 mL, 33.16 mmol, 1.00 equiv) was added dropwise and the mixture was allowed to stir at room temperature for 10 minutes. Following this time, 2-ethyl-2-oxazoline (60.15 g, 605.3 mmol, 18.30 equiv) was added and the resulting mixture was allowed to stir at room temperature for 10 minutes. The reaction was then warmed to 110° C. and stirred for 35 minutes. A separate oven-dried 1000 mL round bottom flask was charged with PhCl (300 mL), followed by NaH (3.97 g, 60% dispersion in oil, 99.22 mmol, 3.00 equiv). Methyl-3-mercaptopropionate (22.00 mL, 198.42 mmol, 6.00 equiv) was then added dropwise and the mixture was allowed to stir for at least 5 hours before use. The polymerization mixture was then cooled to room temperature and subsequently transferred to the termination mixture over 10 minutes under an argon atmosphere. The reaction mixture was allowed to stir at room temperature for at least 12 hours. Following this time, the reaction mixture was concentrated under reduced pressure. 0.1M NaOH (水溶液) (1000 mL) was added and the mixture was stirred for 2 hours. The resulting aqueous solution was passed through a mixed column of Amberlite IRA-67 (200 g) and Amberlite IR120H (200 grams). The product acid was purified via DEAE Sepharose chromatography, the resulting aqueous solution was acidified to pH=3, extracted with CHCl (2×300 mL), and the combined organic extracts were dried over sodium sulfate and concentrated to a volume of 300 mL. The solution was then precipitated into a 4 L beaker containing 3500 mL of EtO, and the solids were collected by vacuum filtration and dried under vacuum to give 46.2 grams of product. 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). A signal for a pendant group was present at δ 1.65 ppm (CH2). The number of pendant groups was calculated via comparison of the integrals of the polymer backbone and the pendant group signals. In this case, the number of pendant groups was determined to be 1.21.

[0259] Example 12. Synthesis of compound 1a

[0260] [ka]

[0261] An oven-dried 500 mL round bottom flask was charged under an argon atmosphere with PhCl (300 mL), followed by a stir bar and 2-(5-pentynyl)-2-oxazoline (2.70 mL, 19.85 mmol, 1.20 equiv). TfOH (1.46 mL, 16.54 mmol, 1.00 equiv) was added dropwise and the mixture was allowed to stir at room temperature for 10 minutes. Following this time, 2-ethyl-2-oxazoline (30.0 g, 302.63 mmol, 18.30 equiv) was added and the resulting mixture was allowed to stir at room temperature for 10 minutes. The reaction was then warmed to 110° C. and stirred for 35 minutes. A separate oven-dried 1000 mL round bottom flask was charged with PhCl (150 mL), followed by NaH (1.98 g, 60% dispersion in oil, 49.61 mmol, 3.00 equiv). Methyl thioglycolate (8.86 mL, 99.24 mmol, 6.00 equiv) was then added dropwise and the mixture was allowed to stir for at least 5 hours before use. The polymerization mixture was then cooled to room temperature and subsequently transferred to the termination mixture over 10 minutes under an argon atmosphere. The reaction mixture was allowed to stir at room temperature for at least 12 hours. Following this time, the reaction mixture was concentrated under reduced pressure. 0.1M NaOH (水溶液)(1000 mL) was added and the mixture was stirred for 2 hours. The resulting aqueous solution was passed through a mixed column of Amberlite IRA-67 (200 g) and Amberlite IR120H (200 grams). The product acid was purified via DEAE Sepharose chromatography, the resulting aqueous solution was acidified to pH=3, extracted with CHCl (2×300 mL), and the combined organic extracts were dried over sodium sulfate and concentrated to a volume of 300 mL. The solution was then precipitated into a 4 L beaker containing 3500 mL of EtO, and the solid was collected by vacuum filtration and dried under vacuum to give 27.6 grams of product. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). It was concluded that the α-methylene of the terminal group was below the signal of the polymer backbone. A signal of the pendant group was present at δ 1.65 ppm (CH2). The number of pendant groups was calculated via comparison of the integral of the polymer backbone and the signal of the pendant group. In this case, the number of pendant groups was determined to be 1.32.

[0262] Example 13. Synthesis of compound 3a

[0263] [ka]

[0264] An oven-dried 500 mL round bottom flask was charged with 2-ethyl-2-oxazoline (32.14 g, 324.22 mmol, 19.00 equiv), followed by PhCl (150 mL) and a stir bar under an argon atmosphere. TfOH (1.51 mL, 17.06 mmol, 1.00 equiv) was added dropwise and the mixture was allowed to stir at room temperature for 5 min. The reaction was then warmed to 110° C. and stirred for 35 min. A separate oven-dried 1000 mL round bottom flask was charged with PhCl (150 mL) followed by NaH (1.91 g, 60% dispersion in oil, 47.8 mmol, 3.00 equiv). Methyl thioglycolate (8.55 mL, 95.60 mmol, 6.00 equiv) was then added dropwise and the mixture was allowed to stir for at least 5 h before use. The polymerization mixture was then cooled to room temperature and subsequently transferred to the termination mixture under an argon atmosphere over 10 minutes. The reaction mixture was allowed to stir at room temperature for at least 12 hours. Following this time, the reaction mixture was concentrated under reduced pressure. 0.1M NaOH (水溶液) (1000 mL) was added and the mixture was stirred for 2 hours. The resulting aqueous solution was passed through a mixed column of Amberlite IRA-67 (200 g) and Amberlite IR120H (200 grams). The product acid was purified via DEAE Sepharose chromatography, the resulting aqueous solution was acidified to pH=3, extracted with CHCl (2×300 mL), and the combined organic extracts were dried over sodium sulfate and concentrated to a volume of 300 mL. The solution was then precipitated into a 4 L beaker containing 3500 mL of EtO, and the solid was collected by vacuum filtration and dried under vacuum to give 8.0 grams of product. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). It was concluded that the α-methylene of the terminal group is located below the signal of the polymer backbone.

[0265] Example 14. Synthesis of Compound 4

[0266] [ka]

[0267] An oven-dried 500 mL round bottom flask was charged with PhCl (300 mL) under an argon atmosphere, followed by a stir bar and 2-(5-pentynyl)-2-oxazoline (5.47 mL, 40.27 mmol, 1.20 equiv). TfOH (2.97 mL, 33.56 mmol, 1.00 equiv) was added dropwise and the mixture was allowed to stir at room temperature for 10 min. Following this time, 2-ethyl-2-oxazoline (60.88 g, 614.14 mmol, 18.30 equiv) was added and the resulting mixture was allowed to stir at room temperature for 10 min. The reaction was then warmed to 110° C. and stirred for 35 min. The polymerization mixture was then cooled to room temperature and a solution of Na2CO3 (22.5 g, 212.3 mmol, 7.00 equiv) in H2O (400 mL) was added. The reaction mixture was allowed to stir for at least 12 h. Following this time, the reaction mixture was transferred to a separatory funnel and diluted with brine (400 mL). The resulting mixture was extracted with CHCl (2×300 mL) and the combined organic extracts were concentrated to a volume of 300 mL. The solution was then precipitated into a 4 L beaker containing 3500 mL of EtO and the solids were collected by vacuum filtration and dried under vacuum to give 57 grams of product. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 4.83-4.65 (CH2CH2OH terminal); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). A signal for pendant groups was present at δ 1.65 ppm (CH2). The number of pendant groups was previously calculated to be 1.13 from the polymer starting material.

[0268] Example 15. Synthesis of Compound 5

[0269] [ka]

[0270] An oven-dried 500 mL round bottom flask was charged with 2-ethyl-2-oxazoline (30.46 g, 307.3 mmol, 19.00 equiv) under an argon atmosphere, followed by PhCl (150 mL) and a stir bar. TfOH (1.43 mL, 16.17 mmol, 1.00 equiv) was added dropwise and the mixture was allowed to stir at room temperature for 5 min. The reaction was then warmed to 110° C. and stirred for 35 min. The polymerization mixture was then cooled to room temperature and ethyl isonipecotate (4.98 mL, 32.34 mmol, 2.00 equiv) was added. The reaction mixture was allowed to stir at room temperature for at least 12 h. Following this time, the reaction mixture was transferred to a separatory funnel and diluted with brine (400 mL). The resulting mixture was extracted with CHCl (2×300 mL) and the combined organic extracts were concentrated to a volume of 300 mL. The solution was then precipitated into a 4 L beaker containing 3500 mL of diethyl ether and the solids were collected by vacuum filtration and dried under vacuum. 25 grams were isolated in this repetition. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 terminal NH); 4.05 (ethyl ester CH2); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 2.81 (CH2-N); 2.32-2.27 (C(O)-CH2); 2.04 (terminal piperidine CH); 1.78 (terminal piperidine CH2); 1.50 (terminal piperidine CH2); 1.16 (ethyl ester CH3); 0.96(CH3). From the material recovered above, 6.00 grams of the ethyl ester was transferred to a 250 mL round bottom flask and diluted with 1M NaOH. (水溶液)(30 mL) was added. The reaction mixture was stirred at room temperature for 12 hours at which point the mixture was acidified to pH=3. The water was removed via rotary evaporation and the residue was dissolved in DMF, filtered, dried over sodium sulfate, and concentrated in vacuo. The resulting gel was dissolved in CH2Cl2, dried over sodium sulfate, and concentrated in vacuo. The product was then precipitated into a beaker containing 2000 mL of diethyl ether. The solid was collected by vacuum filtration and dried under vacuum. 5.80 grams of material was isolated. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18(N-CH2); 2.96 (CH2-N); 2.32-2.27 (C(O)-CH2); 2.06 (terminal piperidine CH); 1.86 (terminal piperidine CH2); 0.96(CH3).

[0271] Example 16. Synthesis of Compound 7

[0272] [ka]

[0273] An oven-dried 500 mL round bottom flask was charged with PhCl (150 mL) under an argon atmosphere, followed by a stir bar and 2-(5-pentynyl)-2-oxazoline (2.7 mL, 19.85 mmol, 1.20 equiv). TfOH (1.46 mL, 16.54 mmol, 1.00 equiv) was added dropwise and the mixture was allowed to stir at room temperature for 10 minutes. Following this time, 2-ethyl-2-oxazoline (30.00 g, 302.6 mmol, 18.30 equiv) was added and the resulting mixture was allowed to stir at room temperature for 10 minutes. The reaction was then warmed to 110° C. and stirred for 35 minutes. The polymerization mixture was then cooled to room temperature. Ethyl isonipecotate (4.98 mL, 33.08 mmol, 2.00 equiv) was then added. The reaction mixture was allowed to stir at room temperature for at least 12 hours. Following this time, the reaction mixture was transferred to a separatory funnel and diluted with brine (400 mL). The resulting mixture was extracted with CH2Cl2 (2 x 300 mL) and the combined organic extracts were concentrated to a volume of 300 mL. The solution was then precipitated into a 4 L beaker containing 3500 mL of Et2O and the solids were collected by vacuum filtration and dried under vacuum. 25 grams of material was isolated. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 terminal NH); 4.05 (ethyl ester CH2); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 2.81 (CH2-N); 2.32-2.27 (C(O)-CH2); 2.04 (terminal piperidine CH); 1.78 (terminal piperidine CH2); 1.50 (terminal piperidine CH2); 1.16 (ethyl ester CH3); 0.96 (CH3). A signal for the pendant group was present at δ 1.65 ppm (CH2). The number of pendant groups was previously calculated to be 0.9 from the polymer starting material.

[0274] Example 17. Synthesis of Compound 8

[0275] [ka]

[0276] An oven-dried 500 mL round bottom flask was charged with chlorobenzene (150 mL) under an argon atmosphere, followed by a stir bar and 2-ethyl-2-oxazoline (30 g, 302.63 mmol, 19.00 equiv). Propargyl tosylate (2.76 mL, 15.93 mmol, 1.0 equiv) was added and the reaction mixture was warmed to 110° C. and stirred for 40 min. A separate oven-dried 1000 mL round bottom flask was charged with chlorobenzene (150 mL), followed by NaH (1.91 g, 60% dispersion in oil, 47.79 mmol, 3.00 equiv). Methyl-3-mercaptopropionate (10.48 mL, 95.58 mmol, 6.00 equiv) was then added dropwise and the mixture was allowed to stir for 5 h. The polymerization mixture was then cooled to room temperature and held for 40 minutes before being transferred to the termination mixture over 10 minutes under an argon atmosphere. The reaction mixture was allowed to stir at room temperature for 12 hours. Following this time, the reaction mixture was concentrated under reduced pressure. 0.1M NaOH (水溶液) (1000 mL) was added and the mixture was stirred for 2 hours. The resulting aqueous solution was passed through a mixed column of Amberlite IRA-67 (200 g) and Amberlite IR120H (200 grams). The product acid was purified via DEAE Sepharose chromatography, the resulting aqueous solution was acidified to pH 3, extracted with CHCl (2×300 mL), and the combined organic extracts were dried over sodium sulfate and concentrated to a volume of 300 mL. The solution was then precipitated into a 4 L beaker containing 3500 mL of EtO, and the solids were collected by vacuum filtration and dried under vacuum to give 15.2 grams of product. HPLC analysis determined analytical purity of 98%. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). Signals of alkynyl groups were present at δ 4.22-3.82 ppm (CH2 and CH).

[0277] Example 18. Synthesis of Compound 9

[0278] [ka]

[0279] An oven-dried 500 mL round-bottom flask was charged with PhCl (75 mL) under an argon atmosphere, followed by a stir bar and 2-ethyl-2-oxazoline (15.41 g, 155.47 mmol, 20.00 equiv). Propargyl tosylate (1.34 mL, 7.77 mmol, 1.0 equiv) was added and the reaction mixture was warmed to 110° C. and stirred for 1 h. The reaction mixture was then cooled to room temperature and an aqueous solution of Na2CO3 (6.00 g, 57.00 mmol, 7.00 equiv) in H2O (100 mL) was added. The reaction mixture was allowed to stir for at least 12 h. Following this time, the reaction mixture was transferred to a separatory funnel and diluted with brine (200 mL). The resulting mixture was extracted with CHCl2 (2×200 mL) and the combined organic extracts were concentrated to a volume of 15000 mL. The solution was then precipitated into a 4 L beaker containing 2000 mL of Et2O, and the solid was collected by vacuum filtration and dried in vacuum to give 6.05 grams of product. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 4.83-4.65 (CH2CH2OH end); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). Signals for the alkynyl groups were present at δ 4.17 ppm (CH2 and CH).

[0280] Example 19. Synthesis of Compound 10

[0281] [ka]

[0282] Poly(ethyloxazoline) (OH terminated, 0.5 grams, 0.27 mmol, 1.00 equiv.) was dissolved in 30 mL of THF and transferred to an oven-dried 250 mL round-bottom flask. Potassium tert-butoxide (0.09 g, 0.81 mmol, 3.00 equiv.) was added and the reaction mixture was allowed to stir at room temperature for 30 minutes. Following this time, tert-butyl bromoacetate (0.24 mL, 1.62 mmol, 6.00 equiv.) was added and the reaction mixture was allowed to stir at room temperature for at least 12 hours. Following this time, the reaction mixture was transferred to a separatory funnel with brine (50 mL). The mixture was extracted with CHCl (2×25 mL) and the combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The resulting solid was dissolved in 1 M aqueous NaOH (20 mL) and stirred at room temperature for at least 12 hours. The solution was then acidified to pH=3 and diluted with CHCl. 2( The combined organic extracts were dried over sodium sulfate and concentrated in vacuo to give 0.4 grams of the intermediate carboxylic acid, which was used as is without further purification.

[0283] A 100 mL round bottom flask was charged with the above carboxylic acid (1 g, 0.45 mmol, 1.00 eq.), then CHCl (30 mL), DMAP (0.01 g), and finally NHS (0.04 grams, 0.36 mmol, 2.00 eq.). DCC (0.08 g, 036 mmol, 2.00 eq.) was then added, and the reaction mixture was allowed to stir at room temperature for at least 12 hours. The resulting mixture was then filtered and concentrated to give the intermediate NHS ester, which was used as is without further purification. The NHS ester was then transferred to a 100 mL round bottom flask, and CHCl (30 mL) was added. Dimyristylamine (0.56 grams, 1.36 mmol, 3.00 eq.) was added, followed by triethylamine (0.20 mL, 1.36 mmol, 3.00 eq.). The reaction mixture was stirred for at least 12 hours, at which point the mixture was diluted with brine (20 mL) and transferred to a separatory funnel. The mixture was extracted with CH2Cl2 (2x20 mL) and the combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The product was purified by reverse phase chromatography (C18, acetonitrile:methanol) to give 0.27 grams of the title compound. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 4.27-3.93 (CHO and O-CH-N); 3.35 (CHCH backbone); 2.96 (N-CH); 2.32-2.27 (C(O)-CH); 0.96 (CH). Additional signals for the lipid moiety were present at δ 1.49-1.54 (N-(CH)); 1.27 (CH); 0.87 (CH).

[0284] Example 20. Synthesis of compound 11a

[0285] [ka]

[0286] A 100 mL round bottom flask was charged with the acid described above (0.68 g, 0.31 mmol, 1.00 equiv), then the azidoacetate (0.27 g, 0.46 mmol, 1.50 equiv), THF (50 mL), CuI (0.24 g, 1.24 mmol, 4.00 equiv), and finally triethylamine (0.64 mL, 4.60 mmol, 10.0 equiv). The resulting mixture was heated to 50° C. and stirred overnight. Following this time, the reaction mixture was cooled to room temperature and quenched by the addition of 0.1 M HCl (30 mL) and stirred for 10 min. The resulting mixture was passed through a pre-prepared 60 mL Dowex column. The resulting solution was concentrated on a rotary evaporator at 35° C. and subsequently transferred to a separatory funnel. Brine (20 mL) was added and the mixture was extracted three times with 20 mL portions of DCM. The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The product was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (0.18 grams). 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). Additional signals for the lipid derivative moiety were present at δ 5.10 (CH); 4.26 (CH2); 4.08 (CH2); 1.49 (C(O)CH2); 1.23 (CH2); 0.84 (CH3). Additional signals for the triazole moiety were present at δ 5.34 (triazole CH); 4.35 (ester CH2); 4.11 (HN-CH2).

[0287] Example 21. Synthesis of compound 11b

[0288] [ka]

[0289] To a solution of Ptyn-PEOZ 2K acid (0.500 g, 0.227 mmol, 1.0 equiv, Mn 2199 Da) and DMG-(2-azidopropionate (0.166 g, 0.273 mmol, 1.2 equiv) in THF (10 mL) was added CuI (0.0217 g, 0.114 mmol, 0.5 equiv) and TEA (0.0634 mL, 0.455 mmol, 2.0 equiv). The resulting mixture was stirred at room temperature for 5 min and then allowed to stir at 50° C. for 18 h, resulting in a cloudy yellow solution. After cooling to room temperature, the reaction mixture was quenched by adding 0.1 N aqueous HCl (6 mL) followed by stirring for 5 min. The mixture was purified by Dowex® The filtrate was passed through a M4195 column and then THF was removed from the filtrate using a rotary evaporator. The resulting aqueous solution was stirred with 20 mL of dichloromethane using 0.5 g of NaCl (5 w / v% of the water volume). The organic phase was collected, dried over Na2SO4, filtered, and concentrated. The residue was dissolved in 5 mL of DCM and precipitated by adding to diethyl ether (60 mL), filtered, and dried in vacuum. The resulting pale yellow crystals were further purified using Biotage (SNAP ultra C-18 column, MeCN / MeOH) to remove polymeric impurities. Fractions 5-21 were collected and concentrated to give the desired product (0.20 g, 31.3% yield with 99.8% purity) as white crystals.

[0290] The DMG-(2-azidopropionate) bond, in addition to the usual polymer backbone peaks, also exhibits DMG protons at 0.84 ppm (t, 6H, J = 6.5 Hz, -(CH2) 10 CH3), 1.23ppm(m, 40H, -(CH2) 10 CH3), 1.49ppm(m, 4H, -CH2(CH2) 10 CH3), 1.81 ppm (d, 3H, J = 5.0 Hz, triazole -CH(CH3)C(=O)O-), 2.28 ppm (m, 4H, -CH2CH2(CH2) 10CH3), 3.07 ppm, 4.23 ppm and 4.33 ppm (m, 4H, -OCH2CH(O-)CHO-), 5.18 ppm (m, 1H, -OCH2CH(O-)CHO-), 5.59 ppm (m, 1H, triazole-CH(CH3)C(=O)O-), and 8.09 ppm (s, 1H, triazole ring, due to the "click" reaction). 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6) spectrum.

[0291] Example 22. Synthesis of compound 11c

[0292] [ka]

[0293] A 100 mL round bottom flask was charged with the acid described above (1.42 g, 0.65 mmol, 1.00 equiv), then the azidopropionate (0.59 g, 0.97 mmol, 1.50 equiv), THF (50 mL), CuI (0.5 g, 2.6 mmol, 4.00 equiv), and finally triethylamine (0.91 mL, 6.50 mmol, 10.0 equiv). The resulting mixture was heated to 50° C. and stirred overnight. Following this time, the reaction mixture was cooled to room temperature and quenched by the addition of 0.1 M HCl (30 mL) and stirred for 10 min. The resulting mixture was passed through a pre-prepared 60 mL Dowex column. The resulting solution was concentrated on a rotary evaporator at 35° C. and subsequently transferred to a separatory funnel. Brine (20 mL) was added and the mixture was extracted three times with 20 mL portions of DCM. The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The product was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (0.34 grams). 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). Additional signals for the lipid derivative moiety were present at δ 5.10 (CH); 4.51 (CH2); 4.26 (CH2); 4.08 (CH2); 1.49 (C(O)CH2); 1.23 (CH2); 0.84 (CH3). Additional signals for the triazole moiety were present at δ 4.35 (ester CH2); 4.11 (HN-CH2).

[0294] Example 23. Synthesis of compound 12a

[0295] [ka]

[0296] A 100 mL round bottom flask was charged with the acid described above (0.82 g, 0.37 mmol, 1.00 equiv), then the azidoacetate (0.40 g, 0.67 mmol, 1.80 equiv), THF (50 mL), CuI (0.28 g, 1.48 mmol, 4.00 equiv), and finally triethylamine (0.52 mL, 3.70 mmol, 10.0 equiv). The resulting mixture was heated to 50° C. and stirred overnight. Following this time, the reaction mixture was cooled to room temperature and quenched by the addition of 0.1 M HCl (30 mL) and stirred for 10 min. The resulting mixture was passed through a pre-prepared 60 mL Dowex column. The resulting solution was concentrated on a rotary evaporator at 35° C. and subsequently transferred to a separatory funnel. Brine (20 mL) was added and the mixture was extracted three times with 20 mL portions of DCM. The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The product was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (0.22 grams). 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). Additional signals for the lipid derivative moiety were present at δ 5.20 (CH); 4.26 (CH2); 1.49 (C(O)CH2); 1.23 (CH2); 0.84 (CH3). Additional signals for the triazole moiety were present at δ 5.35 (ester CH2); 4.11 (HN-CH2).

[0297] Example 24. Synthesis of compound 19b A. Synthesis of DMG-(2-azidopropionate)

[0298] [ka]

[0299] To a solution of DMG (1.02 g, 1.900 mmol, 1.0 equiv) in toluene (20 mL) was added 2-azidopropionic acid (0.289 g, 2.280 mmol, 1.2 equiv, 90.8 wt%) and DMAP (0.0232 g, 0.019 mmol, 0.1 equiv). After adding DCC (0.470 g, 2.280 mmol, 1.2 equiv), the resulting mixture was allowed to stir at room temperature for 2 h. The resulting mixture was filtered using a syringe filter and concentrated using a rotary evaporator. The residue was purified using Biotage (SNAP ultra-silica gel column, EtOAc / Hex) to give the desired product (1.09 g, 93.9% yield, 99.9% purity).

[0300] The bond of 2-azidopropionic acid transfers the proton to 0.85 ppm (t, 6H, J = 6.5 Hz, -(CH2) 10 CH3), 1.23ppm(m, 40H, -(CH2) 10 CH3), 1.32ppm(m, 3H, N3CH(CH3)C(=O)O-), 1.50ppm(m, 4H, -CH2(CH2)10 CH3), 2.28ppm(m, 4H, -CH2CH2(CH2) 10 CH3), 4.15 ppm and 4.35 ppm (m, 4H, -OCH2CH(O-)CH2O-), 4.29 ppm (m, 1H, N3CH(CH3)C(=O)O-), and 5.24 ppm (m, 1H, -OCH2CH(O-)CH2O-). 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6) spectrum.

[0301] B. Synthesis of PEOZ 2K DMG-(triazole-2-propionate)

[0302] [ka]

[0303] To a solution of PEOZ 2K acetylene (1.01 g, 0.450 mmol, 1.0 equiv, Mn 2249 Da) and DMG-(2-azidopropionate (0.302 g, 0.495 mmol, 1.1 equiv) in THF (10 mL) was added CuI (0.0429 g, 0.225 mmol, 0.5 equiv) and TEA (0.125 mL, 0.900 mmol, 2 equiv). The resulting mixture was stirred at room temperature for 4 min and then allowed to stir at 50° C. for 18 h. After cooling to room temperature, the reaction mixture was quenched by the addition of 0.1 N aqueous HCl (12 mL) followed by stirring for 5 min. The mixture was immersed in Dowex® The filtrate was passed through a M4195 column and THF was removed from the filtrate using a rotary evaporator. The resulting aqueous solution was stirred with 30 mL of dichloromethane using 2 g of NaCl (10 w / v% of the water volume). The organic phase was collected, dried over Na2SO4, filtered, and concentrated. The residue was dissolved in 6 mL of DCM and precipitated by adding to diethyl ether (70 mL), filtered, and dried in vacuum. The resulting pale yellow crystals were further purified using Biotage (SNAP ultra C-18 column, MeCN / MeOH) to remove polymeric impurities, followed by lyophilization to give the desired product (0.560 g, 43.8% yield with 99.8% purity) as white crystals.

[0304] The DMG-(2-azidopropionate) bond, in addition to the usual polymer backbone peaks, also exhibits DMG protons at 0.84 ppm (t, 6H, J = 6.5 Hz, -(CH2) 10 CH3), 1.23ppm(m, 40H, -(CH2) 10 CH3), 1.49ppm(m, 4H, -CH2(CH2) 10 CH3), 1.71 ppm (d, 3H, J = 7.5 Hz, triazole -CH(CH3)C(=O)O-), 2.28 ppm (m, 4H, -CH2CH2(CH2) 10CH3), 4.06 ppm and 4.28 ppm (m, 4H, -OCH2CH(O-)CHO-), 5.18 ppm (m, 1H, -OCH2CH(O-)CHO-), 5.64 ppm (m, 1H, triazole-CH(CH3)C(=O)O-), and 8.00 ppm (s, 1H, triazole ring, obtained by "click" reaction). 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6) spectrum.

[0305] Example 25. Synthesis of compound 19c Step 1 – 1,2-Dimyristoyl-3-azidopropionyl-rac-glycerol Synthesis of (DMG-3-azidopropionate)

[0306] [ka]

[0307] 1,2-Dimyristoyl-rac-glycerol (DMG, 1.00 gm, 1.95 mmol, 1.0 equiv) was dissolved in anhydrous DCM (50 mL). Under argon, anhydrous pyridine (632 μL, 7.80 mmol, 4.0 equiv) was added followed by 3-azidopropionyl chloride (521 mg, 3.90 mmol, 2.0 equiv). The reaction mixture was allowed to stir at room temperature under argon. After reacting for 1 h, the solution was evaporated to dryness. The residue was dissolved in DCM (100 mL) and it was washed with 0.05 M HCl (2×40 mL). The DCM phase was dried over anhydrous MgSO4 (4 g). The mixture was filtered. The clear filtrate was evaporated to dryness to give 1.228 g of crude product (yellow liquid). The crude product was purified by flash chromatography on a Biotage SNAP Ultra 25 g column, Biotage Isolera System, using hexane and ethyl acetate as the mobile phase. After column purification, the mobile phase of the product fraction was evaporated and the residue was further dried in vacuum overnight to give 0.72 g of a colorless liquid / white wax. TLC (silica gel 60) shows one spot. 1H NMR (Varian, 500 MHZ, 4 mg / mL DMSO-d6, δ, ppm, TMS): 0.85 (t, 2 × 3H, -CH3), 1.23 (m, poorly resolved, 2 × 20H, -(CH2) 10 -), 1.50 (m, 2 × 2H, -(C=O)CH2CH2-), 2.28 (t, 2 × 2H, -(C=O)CH2-), 2.61 (t, 2H, N3CH2CH2-), 3.54 (t, 2H, N3CH2-), 4.14, 4.20 (qq, 2H, -CH2O(C=O)C 13 H 27 ), 4.28 (d, 2H, -CH2O(C=O)C2H4N3), 5.20 (m, 1H, -(OCH2)2CH-O-).

[0308] Step 2 - Synthesis of PEOZ-(triazole-3-propionyl-DMG) 2K, 19c via click reaction

[0309] [ka]

[0310] H-PEOZ-propargylamide 2K (1.0 gm, 0.67 mmol, 1 eq) was dissolved in 25 mL of THF in a 50 mL round bottom flask along with DMG-3-azidopropionate (0.464 gm, 0.76 mmol, 1.1 eq). The solution was stirred under a slow argon flow. CuI (72.4 mg, 0.38 mmol, 0.5 eq) was then added to the flask followed by TEA (185.4 μL, 1.33 mmol, 1.9 eq). The greenish solution was left stirring overnight at 50° C. in an oil bath under an argon atmosphere. The reaction was complete as indicated by reverse phase HPLC analysis of the reaction mixture. The solution was mixed with 26.6 mL of 50 mM HCl (1.33 mmol) and THF (25 mL). The copper in the solution was removed by passing the solution through Ambersep M4195 media packed in a glass column. The column was eluted with THF-2 mM HCl (2:1 (v / v)). The eluent (150 mL) was evaporated until the THF was removed. NaCl (5 g) was added to the remaining white cloudy aqueous solution and extracted with DCM (4 times x 50 mL). The DCM phase was dried with anhydrous magnesium sulfate (3 g) and anhydrous sodium sulfate (20 g). After filtering to remove magnesium sulfate and sodium sulfate, the filtrate was concentrated to near dryness and then redissolved in 4 mL of DCM and then precipitated in diethyl ether (160 mL). After filtering, the precipitate was collected and dried in vacuum to give 0.87 g of amber powder (PEOZ-(triazole-3-propionyl-DMG)2K). The purity by reverse phase HPLC is 99.7%. 1 H NMR (Varian, 500 MHZ, 10 mg / mL DMSO-d6, δ, ppm, TMS): 0.85 (t, 2 × 3H, -C 13 H 24 CH3), 1.23 (m, poorly resolved, 2 × 20H, -(CH2) 10 -), 1.49 (m, 2 × 2H, -(C=O)CH2CH2-), 2.04 (t, 2 × 2H, -(C=O)CH2-), 2.97 (t, 2H, -triazole-CH2CH2-), 4.10, 4.16 (mm, 2H, -CH2O(C=O)C13 H 27 ), 4.28 (d, 2H, -CH2O(C=O)C2H4-triazole), 4.54 (t, 2H, -triazole-CH2-), 5.17 (m, 1H, -(OCH2)2CH-O-), 7.90 (s, 1H, -CH- on the triazole ring), 8.51 (t, poorly resolved, 1H, -(C=O)NH-CH2-triazole-). The PEOZ backbone peaks are at 0.95 (medium) (s, 3nH, CH3CH2(C=O)NH-), 2.27 (small) (s, 2nH, -CH2(C=O)NH-), and 3.35 (large) (s, 4nH, -NCH2CH2N-).

[0311] Example 26. Synthesis of compound 20a

[0312] [ka] Synthesis of PMOZ 2K DMG ester To a solution of PMOZ 2K acid (1.08 g, 0.500 mmol, 1.0 equiv, Mn 2166 Da) in DCM (12 mL) was added 1,2-dimyristoyl-rac-glycerol (0.283 g, 0.525 mmol, 1.05 equiv, 95%) and DMAP (0.0062 g, 0.050 mmol, 0.1 equiv). At 45° C., DCC (0.108 g, 0.525 mmol, 1.05 equiv) was added and the resulting mixture was allowed to stir for 1 h. To the mixture was added fresh additional DMG (0.283 g, 0.525 mmol, 1.05 equiv, 95%) and DCC (0.108 g, 0.525 mmol, 1.05 equiv). After stirring at 45° C. for 16 h, the reaction mixture was cooled to room temperature, filtered using a syringe filter, and concentrated using a rotary evaporator. The residue was purified using Biotage (SNAP ultra C18 column, MeCN / MeOH) to give the desired product (0.40 g, 30% yield, 99.8% purity).

[0313] The DMG bond is characterized by the usual polymer backbone peaks, as well as the DMG protons at 0.85 ppm (t, 6H, J = 6.5 Hz, -(CH2) 10 CH3), 1.23ppm(m, 40H, -(CH2) 10 CH3), 1.50ppm(m, 4H, -CH2(CH2) 10 CH3), 2.72ppm(t, 4H, J=6.5 Hz, - CH2CH2(CH2) 10 CH3), 4.15 ppm and 4.27 ppm (m, 2H, -OCH2CH(O-)CH2O-), and 5.19 ppm (m, 1H, -OCH2CH(O-)CH2O-). 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6) spectrum.

[0314] Example 27. Synthesis of compound 20b

[0315] [ka]

[0316] Synthesis of PEOZ 2K DMG ester To a solution of PEOZ 2K acid (0.660 g, 0.300 mmol, 1.0 equiv, Mn 2200 Da) in DCM (12 mL) was added 1,2-dimyristoyl-rac-glycerol (0.170 g, 0.315 mmol, 1.05 equiv, 95%) and DMAP (0.0037 g, 0.030 mmol, 0.1 equiv). After addition of DCC (0.065 g, 0.315 mmol, 1.05 equiv), the resulting mixture was allowed to stir for 18 h. The mixture was filtered using a syringe filter and concentrated using a rotary evaporator. The residue was purified using Biotage (SNAP ultra C18 column, MeCN / MeOH) to give the desired product (0.624 g, 78% yield, >99.9% purity).

[0317] The DMG bond is characterized by the usual polymer backbone peaks, as well as the DMG protons at 0.85 ppm (t, 6H, J = 6.5 Hz, -(CH2)10 CH3), 1.23ppm(m, 40H, -(CH2) 10 CH3), 1.50ppm(m, 4H, -CH2(CH2) 10 CH3), 2.70ppm(t, 4H, J=6.5Hz, -CH2CH2(CH2) 10 CH3), 4.15 ppm and 4.26 ppm (m, 2H, -OCH2CH(O-)CH2O-), and 5.19 ppm (m, 1H, -OCH2CH(O-)CH2O-). 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6) spectrum.

[0318] Example 28. Synthesis of compound 21

[0319] [ka]

[0320] To a solution of Ptyn-PEOZ 2K isonipecotate (compound 7, 0.500 g, 0.274 mmol, 1.0 equiv, Mn 1823 Da) and DMG-(2-azidopropionate (0.277 g, 0.455 mmol, 1.6 equiv) in THF (15 mL) was added CuI (0.0433 g, 0.227 mmol, 0.83 equiv) and TEA (0.095 mL, 0.682 mmol, 2.5 equiv). The resulting mixture was stirred at room temperature for 5 min and then allowed to stir at 50° C. for 18 h, resulting in a cloudy yellow solution. After cooling to room temperature, the reaction mixture was quenched by adding 0.1 N aqueous HCl (8 mL) followed by stirring for 5 min. The mixture was purified by Dowex® The filtrate was passed through a M4195 column and then THF was removed from the filtrate using a rotary evaporator. The resulting aqueous solution was stirred with 20 mL of dichloromethane using 0.5 g of NaCl (5 w / v% of the water volume). The organic phase was collected, dried over Na2SO4, filtered, and concentrated. The residue was dissolved in 5 mL of DCM and precipitated by adding to diethyl ether (60 mL), filtered, and dried in vacuum. The resulting pale yellow crystals were further purified using Biotage (SNAP ultra C-18 column, acetonitrile / methanol) to remove polymeric impurities. Fractions 5-21 were collected and concentrated to give the desired product (0.140 g, 21% yield with 99.8% purity) as a yellow crystalline solid.

[0321] The DMG-(2-azidopropionate) bond, in addition to the usual polymer backbone peaks, also exhibits DMG protons at 0.84 ppm (t, 6H, J = 6.5 Hz, -(CH2) 10 CH3), 1.23ppm(m, 40H, -(CH2) 10 CH3), 1.49ppm(m, 4H, -CH2(CH2) 10 CH3), 1.72 ppm (d, 3H, J = 7.0 Hz, triazole -CH(CH3)C(=O)O-), 2.28 ppm (m, 4H, -CH2CH2(CH2) 10CH3), 4.08 ppm, 4.23 ppm and 4.33 ppm (m, 4H, -OCH2CH(O-)CHO-), 5.18 ppm (m, 1H, -OCH2CH(O-)CHO-), 5.59 ppm (m, 1H, triazole-CH(CH3)C(=O)O-), and 8.00 ppm (s, 1H, triazole ring, obtained by "click" reaction). 1 The reaction was characterized by H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6).

[0322] Example 29. Synthesis of compound 22

[0323] [ka]

[0324] An oven-dried 500 mL round bottom flask was charged with 2-ethyl-2-oxazoline (30 g, 302.63 mmol, 19.00 equiv) under an argon atmosphere, followed by PhCl (150 mL) and a stir bar. TfOH (1.41 mL, 15.93 mmol, 1.00 equiv) was added dropwise and the mixture was allowed to stir at room temperature for 5 minutes. The reaction was then warmed to 110° C. and stirred for 35 minutes. The polymerization mixture was then cooled to room temperature and dimyristylamine (13.00 grams, 31.86 mmol, 2.00 equiv) was added. The reaction mixture was allowed to stir at room temperature for at least 12 hours. Upon completion of this time, the reaction mixture was diluted with brine (300 mL) and transferred to a separatory funnel. The mixture was extracted with CHCl (2×150 mL), dried over sodium sulfate, and concentrated in vacuo to give 25.6 grams of crude material. A 2 gram sample of the crude material was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (0.55 grams). 1 H NMR. 1H NMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 3.42 (CH2CH2 backbone); 3.27 (N-CH2); 2.37-2.25 (C(O)-CH2); 1.11 (CH3). Additional signals for the lipid moiety were present at δ 2.74 (N-(CH2)2); 2.50 (N-CH2); 1.66 (CH2); 1.35 (CH2); 1.22 (lipid alkyl chain); 0.85 (CH3).

[0325] Example 30. Synthesis of compound 13

[0326] [ka]

[0327] Compound 13 was prepared in a manner similar to compound 15B. 1.4 grams was isolated. 1 H NMR. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 3.43 (CH2CH2 backbone); 3.24 (N-CH2); 2.8 (S-CH2); 2.32-2.19 (C(O)-CH2); 1.12 (CH3). Additional signals for the lipid moiety were present at δ 1.49-1.65(N-(CH2)2); 1.27(CH2); 0.87(CH3).

[0328] Example 31. Synthesis of compound 14

[0329] [ka]

[0330] The carboxylic acid (2.00 grams, 0.91 mmol, 1.00 equiv.) was transferred to a 250 mL round bottom flask and azeotroped with acetonitrile. This process was repeated and the residue was dried under vacuum for 1 hour. The residue was dissolved in CHCl (30 mL) and HOBT (0.05 g, 0.36 mmol, 0.4 equiv.) was added, followed by dimyristylamine (1.12 grams, 2.73 mmol, 3.00 equiv.) and finally DCC (0.6 grams, 2.73 mmol, 3.00 equiv.). The reaction mixture was allowed to stir for at least 12 hours whereupon the mixture was filtered and precipitated into a beaker containing 1600 mL of hexane. The solid was collected by vacuum filtration and dried under vacuum. The product was purified by reverse phase C18 chromatography using acetonitrile and methanol as eluents to give the title compound (0.47 grams). 1 H NMR. 1 H NMR analysis showed standard backbone signals for PEOZ (500 MHz, CDCl3) δ 3.42 (CH2CH2 backbone); 2.93 (N-CH2); 2.39-2.27 (C(O)-CH2); 1.11 (CH3). Additional signals for the piperidine moiety were present at δ 2.92 (N-CH); 1.83 (CH2); 1.76 (CH2); 1.74 (CH2); 1.63 (CH2). Additional signals for the lipid moiety were present at δ 3.25 (N-CH2); 3.19 (N-CH2); 2.05 (CH2); 1.52-1.46 (CH2); 1.25 (lipid alkyl chain); 0.86 (CH3).

[0331] Example 32. Synthesis of compound 23

[0332] [ka]

[0333] Compound 23 was prepared in a manner similar to compound 12A. In this case, 0.19 g was isolated. The analytical purity was determined to be 93% by HPLC analysis. 1HNMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 3.35 (CH2CH2 backbone); 2.71 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96(CH3). Additional signals for the lipid derivative moiety were present at δ 5.19(CH); 4.26(CH2); 1.49(C(O)CH2); 1.23(CH2); 0.84(CH3). Additional signals for the triazole moiety were present at δ 5.46 (ester CH2); 4.07 (HN-CH2).

[0334] Example 33 - Preparation of lipid nanoparticles containing N,N-dimethyltetradecylamine (DMA) Fresh lipid stock solutions of 1,2-dioleoyloxy-3-(trimethylammonium)propane [DOTAP] (Sigma D6182), 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC] (Bachem 4005619), cholesterol ultrapure (VWR 0433) and 2K polymer (PEG, PMOZ or PEOZ) conjugated DMA (as described in the above examples) were prepared as stock solutions in ethanol. The stock solutions were mixed so that the final lipid mixture stock contained 1.30 mg / mL DOTAP, 0.28 mg / mL DSPC, 0.59 mg / mL cholesterol and 0.19 mg / mL polymeric DMA. The solution contained 2.38 mg / mL total lipid. Then, 70 μL of the stock solution was pipetted into a 1.5 mL LoBind centrifuge tube (Eppendorf type 022431081) and the ethanol solution was evaporated using a speed vacuum system (GeneVac EZ-2). The dried sample was left to dry for an additional 10 min under a vented snorkel. The dried lipid mix was hydrated with 250 μL of citrate buffer (pH 4.0), mixed, and sonicated for approximately 10 min to prepare lipid nanoparticles (LNPs).

[0335] Plasmid DNA (phMGFP, Promega E6421) stock solution was mixed with citrate buffer (pH 4.0) such that the total volume was 166.7 μL with a ratio of 100 μL of concentration 0.34 μg / μL to 66.7 μL of citrate buffer. A 50 μL aliquot of the plasmid solution containing 10 μg of DNA was added to 250 μL of lipid nanoparticle mixture, pipette mixed, and sonicated for less than 1 min to obtain a translucent-like suspension. The tube was centrifuged at 14,000 rpm for 30 min (Eppendorf microcentrifuge). Approximately 300 μL of the supernatant was separated from the LNP pellet and assayed for any residual DNA using a 1.2% agarose gel and Qubit quantification. Results showed no DNA was present in the supernatant, suggesting complete encapsulation.

[0336] The pellet was resuspended in 300 μL of 5 mM HEPES buffer (pH 7.40) and mixed well with a pipette tip for 2 min. To verify DNA encapsulation of each polymer-DMA formulation, an aliquot of the LNP suspension was dissolved with Triton X-100. First, a 10% Triton stock solution was made in HEPES buffer. 3 μL of this solution was added to 27 μL of LNP suspension such that the final volume was 30 μL and the concentration of Triton X-100 surfactant was 1%. The suspension was mixed by vortexing for 2 min and left at 4 °C overnight. The mixtures were assayed for any residual DNA using a 1.2% agarose gel and Qubit quantification. The results showed a high concentration of DNA in the mixtures, suggesting that the encapsulation efficiency of each LNP formulation was >90%. The suspension is easily filtered through 0.22 and 0.1 μm PVDF low protein binding 33 mm syringe filters (Millex-W and Millex-GV, Millipore).

[0337] Example 34 - Preparation of lipid nanoparticles containing 1,2-dimyristoyl-sn-glycerol (DMG) Fresh lipid stock solutions of 1,2-dioleoyloxy-3-(trimethylammonium)propane [DOTAP] (Sigma D6182), 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC] (Bachem 4005619), cholesterol ultrapure (VWR 0433) and 2K and 5K polymer (PEG, PMOZ or PEOZ) conjugates DMG (described in the above examples) were prepared as stock solutions in ethanol. The stock solutions were mixed so that the final lipid mixture stock contained 1.48 mg / mL DOTAP, 0.28 mg / mL DSPC, 0.57 mg / mL cholesterol and 0.15 mg / mL 2K polymer DMG. The solution contained 2.49 mg / mL total lipid. When the 5K polymer DMG conjugate was selected, the concentration was 0.32 mg / mL and the solution contained 2.67 mg / mL total lipid. 62 μL of the stock solution was then pipetted into a 1.5 mL LoBind centrifuge tube (Eppendorf type 022431081) and the ethanol solution was evaporated using a speed vacuum system (GeneVac EZ-2). The dried sample was left to dry for an additional 10 min under a vented snorkel. The dried lipid mix was hydrated with 275 μL of citrate buffer (pH 4.0), mixed, and sonicated for approximately 10 min to prepare lipid nanoparticles (LNPs).

[0338] Plasmid DNA (phMGFP, Promega E6421) stock solution was mixed with citrate buffer (pH 4.0) in a ratio of 80 μL of 2.7 μg / μL concentration to 190 μL of citrate buffer in a total volume of 270 μL. A 25 μL aliquot of the plasmid solution containing 20 μg of DNA was added to 275 μL of lipid nanoparticle mixture, pipette mixed, and sonicated for less than 1 min to obtain a translucent-like suspension. The tube was centrifuged at 14,000 rpm for 30 min (Eppendorf microcentrifuge). Approximately 300 μL of the supernatant was separated from the LNP pellet and assayed for any residual DNA using a 1.2% agarose gel and Qubit quantification. Results showed no DNA was present in the supernatant, suggesting complete encapsulation.

[0339] The pellet was resuspended in 300 μL of 5 mM HEPES buffer (pH 7.40) and mixed well with a pipette tip for 2 min. To verify DNA encapsulation of each polymer-DMA formulation, an aliquot of the LNP suspension was dissolved with Triton X-100. First, a 10% Triton stock solution was made in HEPES buffer. 3 μL of this solution was added to 27 μL of LNP suspension such that the final volume was 30 μL and the concentration of Triton X-100 surfactant was 1%. The suspension was mixed by vortexing for 2 min and left at 4 °C overnight. The mixtures were assayed for any residual DNA using a 1.2% agarose gel and Qubit quantification. The results showed a high concentration of DNA in the mixtures, suggesting that the encapsulation efficiency of each LNP formulation was >90%. The suspension is easily filtered through 0.22 and 0.1 μm PVDF low protein binding 33 mm syringe filters (Millex-W and Millex-GV, Millipore).

[0340] In another experiment, the centrifuged pellet was resuspended in a buffer containing sodium dihydrogen phosphate, disodium hydrogen phosphate, and sucrose (pH 7.4). The suspension was lyophilized to give a uniform and stable dry cake that was easily reconstituted in water for injection.

[0341] Example 35. Transfection of DNA from LNPs into HEK-293 cells HEK-293 is a human embryonic kidney cell line commonly used in cell biology for transfection studies. Cells were sourced from ATCC (CRL-1573) and were demonstrated to have a transfection efficiency of >85%. In the study protocol, low passage (<5) cells are seeded at a density of 20,000 cells per well in 100 μL of antibiotic-free medium (EMEM+10% FBS) in assay plates 24 hours prior to transfection. Immediately prior to transfection, a complete medium change is performed and 100 μL of fresh antibiotic-free medium (EMEM+10% FBS) is added to each well.

[0342] Add prepared LNPs containing polymeric DMA lipids and encapsulated plasmid DNA to each well in a volume of 2.6-20.8 µL to deliver 50-450 ng of DNA phMGFP per well.

[0343] Positive controls are FuGENE-HD (Promega E3211) and DNA in a 3:1 ratio. The DNA solution is diluted in serum-free medium (Opti-MEM). In a 96-well PCR plate, both solutions are mixed in a 3:1 ratio (0.3 μL of reagent to 100 ng of plasmid DNA per well) in triplicates and Opti-MEM is added to a volume of 7.33 μL. The plate is incubated at room temperature for 25 minutes. 7.33 μL of the DNA / transfection mix is ​​added to the cells in each well.

[0344] Cover the plates with the lids provided and place them in an incubator at 37°C, 5% CO2, 95% RH. Incubate them for 24-48 hours. At each of the time points, read the plates on an Evos microscope. Read all GFP filter photos at 4x and all transmitted light photos at 10x. The number of cells that fluoresced with GFP are shown below in Table 1. The transmitted light photos showed some pathological cells at the highest concentration tested for all polymer-DMA lipid formulations. Notably, the PEG-DMA lipid had the most cells that fluoresced with GFP.

[0345] [Table 1]

[0346] At the 48 hour time point, fluorescence was high and observations showed that 30-35% of cells fluoresced (positive control); 10% (PMOZ DMA lipid, 466 ng DNA); 10% (PEOZ DMA lipid, 350 ng DNA); and 5% (PEG DMA lipid, 450 ng DNA) were cell dead.

[0347] Example 36. Transfection of DNA from LNPs into HepG2 cells HepG2 is a human hepatocellular carcinoma cell line that is frequently used in transfection studies. The cells were sourced from ATCC (HB-8065) ​​and were demonstrated to have a high transfection efficiency of 95%. In the study protocol, low passage (<5) cells are seeded at a density of 40,000 cells per well in 100 μL of antibiotic-free medium (DMEM + 10% FBS) in assay plates 24 hours prior to transfection. Just prior to transfection, a complete medium change is performed and 100 μL of fresh antibiotic-free medium (DMEM + 10% FBS) is added to each well.

[0348] Add prepared LNPs containing polymeric DMG lipids and encapsulated plasmid DNA to each well in a volume of 1.9-11.7 µL to deliver 100-1000 ng of DNA phMGFP per well.

[0349] Positive controls are FuGENE-HD (Promega E3211) and DNA in a 3:1 ratio. The DNA solution is diluted in serum-free medium (Opti-MEM). In a 96-well PCR plate, both solutions are mixed in a 3:1 ratio (0.3 μL of reagent to 100 ng of plasmid DNA per well) in triplicates and Opti-MEM is added to a volume of 7.33 μL. The plate is incubated at room temperature for 25 minutes. 7.33 μL of the DNA / transfection mix is ​​added to the cells in each well.

[0350] Cover the plates with the lids provided and place them in a 37°C, 5% CO2, 95% RH incubator. Incubate them for 48-72 hours. At each of the time points, the plates are read on an Evos microscope. All GFP filter photographs are read at 4x and all transmitted light photographs are read at 10x. The number of cells that fluoresced GFP is shown below in Table 2. Transmitted light photographs showed no signs of cytotoxicity.

[0351] [Table 2]

[0352] Example 37. Hydrolysis kinetics of POZ-lipid conjugates The hydrolysis rate of some POZ-lipid conjugates of the present disclosure was determined in biological media including phosphate-saline buffer, pH 7.4 (PBS) and 100% or 50% rat and human plasma diluted in PBS. POZ-lipid samples were accurately weighed and dissolved in PBS to prepare stock solutions. These solutions were added to PBS or rat or human plasma media and allowed to incubate at 37°C. Samples were removed from the hydrolysis media at various time intervals and quenched by adding chilled 0.2% formic acid in methanol. Samples were centrifuged and the supernatant transferred to 0.1% formic acid in water, then analyzed by high performance liquid chromatography (HPLC) using a reversed-phase column (300SB C3) and a gradient flow of 0.1% ammonium formate in water and 0.1% ammonium formate in methanol as the mobile phase. POZ-lipid conjugates were detected by UV absorption at 210 nm, and their degradation and degradation metabolites were detected, followed by mass spectrometry. The time taken for 50% of the POZ-lipid to degrade was extrapolated from the concentration-time curves and is reported below in Table 3.

[0353] [Table 3]

[0354] The results show that compounds with ester linkages (compounds 19a, 19b, 19c, 20a and 20b) hydrolyzed faster in plasma than compounds with ether linkages (compounds 17a, 17b and 17c).

[0355] Example 38. Amidase hydrolysis of polymer-lipid conjugates The purpose of this experiment was to determine whether various polymer-lipid conjugates that showed no apparent reaction (hydrolysis) in PBS, rat plasma, or human plasma (examples above) would react in the presence of lipo-amidase enzyme. Compounds 13, 15a, and 15b have an amide linkage between POZ and the lipid. Compound 22 has an amine linkage. POZ-lipid samples were accurately weighed and dissolved in PBS to prepare stock solutions. The amidase selected was "Fatty Acid Amide Hydrolase 1, Active Human Recombinant (FAAH1)" supplied by Bio-Vision, with a specific activity of ≥ 9mU / mg. The amidase was activated by adding PBS pH 8.0 to each vial of amidase, gently mixing, and placing in a 37°C water bath for 2 hours. The concentration of the amidase solution was approximately 2 μg / μL. An aliquot of the POZ-lipid stock solution was added to the activated lipo-amidase solution, mixed gently, and allowed to incubate overnight at 37°C. After overnight hydrolysis, methanol was added, the sample was centrifuged, and the supernatant was transferred to an HPLC vial. The samples were analyzed by high performance liquid chromatography (HPLC) using a reversed-phase column (300SB C3) and a gradient run of 0.1% ammonium formate in water and 0.1% ammonium formate in methanol as the mobile phase. The peak area of ​​the polymer lipid conjugate was measured by UV absorption at 210 nm. The peak areas from the initial and overnight amidase incubations were compared, and the results are shown below in Table 4.

[0356] [Table 4]

[0357] The results show that the compounds with amide linkages (compounds 15a, 15b, and 13) were affected by the lipo-amidase enzyme as observed in the decrease in POZ-DMA peak area after overnight hydrolysis, i.e., a change of 30-50%. In contrast, compound 22 with amine linkage showed a slight decrease in peak area, which was not significant and was within the error range of the experimental conditions, i.e., ±10%.

[0358] Example 39. Synthesis of compound 24 Step 1.

[0359] [ka]

[0360] PEOZ 2K (1.21p) NHS ester was prepared in a manner similar to that previously described. 1 H NMR. HNMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). An additional signal for the NHS moiety was present at δ 2.81 ((CH2)2). The CH2 signal of the alkyne moiety was detected at δ 1.63 ppm.

[0361] Step 2.

[0362] [ka]

[0363] PEOZ 2K (1.21p) ethanolamine glycerol amide was prepared in a manner similar to that previously described. 1 H NMR. HNMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02(S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). The CH2 signal of the alkyne moiety was detected at δ 1.63 ppm. Additional signals for the diol moiety were present at δ 4.56 (CH2); 4.53 (CH).

[0364] Step 3.

[0365] [ka]

[0366] PEOZ 2K (1.21p) DMG was prepared in a manner similar to that previously described. 1 H NMR. HNMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02(S-CH2); 2.32-2.27 (C(O)-CH2); 0.96(CH3). Additional signals for the lipid derivative moiety were present at δ 5.20(CH); 4.26(CH2); 1.49(C(O)CH2); 1.23(CH2); 0.84(CH3). The CH2 signal of the alkyne moiety was detected at δ 1.63 ppm.

[0367] Step 4.

[0368] [ka]

[0369] PEOZ 2K(1.21p)DMG(3-azidopropionate), compound 24, was prepared in a manner similar to that previously described. 1 H NMR. HNMR analysis showed standard backbone signals for PEOZ (500 MHz, DMSO) δ 7.82 (terminal NH); 3.35 (CH2CH2 backbone); 3.18 (N-CH2); 3.02 (S-CH2); 2.32-2.27 (C(O)-CH2); 0.96 (CH3). Additional signals for the lipid derivative moiety were present at δ 5.20 (CH); 4.26 (CH2); 1.49 (C(O)CH2); 1.23 (CH2); 0.84 (CH3). Additional signals for the triazole moiety were present at δ 7.78 (triazole CH); 4.54 (ester CH2).

[0370] The POZ-lipids, LNPs, and pharmaceutical compositions described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, as these embodiments are intended as illustrations of some aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of the disclosure. Indeed, various modifications of the formulas and structures, in addition to those shown and described herein, will become apparent to those skilled in the art from the above description. Such modifications are also intended to fall within the scope of the appended claims. All patents and patent applications cited in the above text are expressly incorporated herein by reference in their entirety. Any section headings herein are provided solely for consistency with the teachings of 37 CFR § 1.77 or other methods of defining structural order. These headings should not be construed as limiting or characterizing the invention described herein.

Claims

1. Compounds of Formula I: R-POZ-Z-L-lipid I wherein R comprises an initiator group; POZ comprises a polyoxazoline polymer; L is -CH 2 CH 2 -G-, where G comprises a linking group that has controllably degradability in a physiological medium; The compound, wherein the lipid comprises an uncharged lipid comprising at least one hydrophobic moiety.

2. POZ is, -{[N(COX)CH ２ CH ２ ] ｏ -[N(COR ２ )CH ２ CH ２ ] ｎ } ａ - wherein X comprises a functional group comprising an alkyne group, a triazole having an attached carboxylic acid, or a combination thereof, o is in the range of 0 to 10, n is in the range of 1 to 1000, and R ２ 2. The compound of claim 1, wherein R comprises hydrogen, a substituted or unsubstituted alkyl, an alkyne-substituted alkyl, or a substituted or unsubstituted aralkyl group; and a is ran, which represents a random copolymer, or block, which represents a block copolymer.

3. The compound of claim 2, wherein POZ has a polydispersity index of about 1.01 to about 1.

20.

4. The compound of claim 2, wherein POZ has a molecular weight of from 500 Daltons to 10,000 Daltons.

5. 2. The compound of claim 1, wherein R comprises hydrogen, a substituted or unsubstituted alkyl, an alkyne-substituted alkyl, a triazole having an attached carboxylic acid, or a substituted or unsubstituted aralkyl group.

6. The compound of claim 1, wherein G comprises an ether, an ester, a carboxylate, a carbonate, a carbamate, an amine, an amide, a disulfide, and combinations thereof.

7. The compound of claim 1 , wherein the lipid comprises two hydrophobic moieties.

8. 2. The compound of claim 1, wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof.

9. 2. The compound of claim 1, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

10. Formula I is as follows: 【Chemistry 1】 【change】 【change】 【change】 wherein m is 1-2, n is in the range of 1-1000, and p is in the range of 1-10.

11. 2. The compound of claim 1, having a hydrolysis rate determined at least in part by L.

12. 2. The compound of claim 1, having a half-life for hydrolysis in 50% human plasma of about 10 minutes or less.

13. 2. The compound of claim 1, having a half-life for hydrolysis in 50% human plasma of about 120 hours or more.

14. Compound of Formula II: Lipid-L １ - (POZ) ｎ ａ -T II wherein The lipid comprises an uncharged lipid comprising at least one hydrophobic moiety; L １ comprises a linking group that is controllably degradable in a physiological medium, POZ has the structure [N(COR ２ ) CH ２ CH ２ wherein R ２ is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, alkyne-substituted alkyl, or an active functional group for each repeat unit of the polyoxazoline polymer; n is in the range of 1 to 1,000; a is ran representing a random copolymer or block representing a block copolymer; The compound wherein T comprises a terminating end group.

15. L １ The compound of claim 14 , wherein comprises ethers, esters, carboxylates, carbonates, carbamates, amines, amides, disulfides, and combinations thereof.

16. L １ The compound of claim 14 , wherein comprises a triazole.

17. 15. The compound of claim 14, wherein T comprises Z-B-Q, where Z comprises S, O, or N, B is an optional linking group, and Q is a portion of a terminating nucleophile.

18. The compound of claim 14 , wherein the lipid comprises two hydrophobic moieties.

19. 15. The compound of claim 14, wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof.

20. 15. The compound of claim 14, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

21. Formula II is as follows: 【Chemistry 2】 【change】 wherein m is 1-2, n is in the range of 1-1000, and o is in the range of 1-5.

22. 15. The compound of claim 14, having a hydrolysis rate determined at least in part by L.

23. 15. The compound of claim 14, having a half-life for hydrolysis in 50% human plasma of about 10 minutes or less.

24. 15. The compound of claim 14, having a half-life for hydrolysis in 50% human plasma of about 120 hours or more.

25. Compound of Formula III: R-(POZ) ｎ ａ -Z-L ２ - Lipid III wherein R comprises an initiator group; POZ has the structure [N(COR ２ ) CH ２ CH ２ wherein R ２ is independently selected from an unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl group, alkyne-substituted alkyl, or active functional group for each repeat unit of the polyoxazoline polymer; n is in the range of 1 to 1,000; a is ran representing a random copolymer or block representing a block copolymer; Z comprises S, O, or N; L ２ is -CH 2 CH 2 -G-, where G comprises a linking group that has controllably degradability in a physiological medium; The compound, wherein the lipid comprises an uncharged lipid comprising at least one hydrophobic group.

26. The compound of claim 25, wherein G comprises an ether, an ester, a carboxylate, a carbonate, a carbamate, an amine, an amide, a disulfide, and combinations thereof.

27. 26. The compound of claim 25, wherein the lipid comprises two hydrophobic moieties.

28. 26. The compound of claim 25, wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof.

29. 26. The compound of claim 25, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

30. 26. The compound of claim 25, wherein R comprises hydrogen or substituted or unsubstituted alkyl and n is in the range of 15 to 35.

31. 26. The compound of claim 25, having a hydrolysis rate determined at least in part by L.

32. 26. The compound of claim 25, having a half-life for hydrolysis in 50% human plasma of about 10 minutes or less.

33. 26. The compound of claim 25, having a half-life for hydrolysis in 50% human plasma of about 120 hours or more.

34. Compound of Formula IV: 【Chemistry 3】 wherein R comprises an initiator group; L ３ comprises a linking group that is controllably degradable in a physiological medium, The lipid comprises an uncharged lipid comprising at least one hydrophobic moiety; n is in the range of 1 to 5; R ２ is independently selected for each repeat unit from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, alkyne-substituted alkyl, or an active functional group; m is in the range of 1 to 100; a is ran representing a random copolymer or block representing a block copolymer; The compound wherein T comprises a terminating end group.

35. L ３ 35. The compound of claim 34, wherein comprises ethers, esters, carboxylates, carbonates, carbamates, amines, amides, disulfides, and combinations thereof.

36. L ３ The compound of claim 34 , wherein comprises a triazole.

37. 35. The compound of claim 34, wherein T comprises Z-B-Q, where Z comprises S, O, or N, B is an optional linking group, and Q is a portion of a terminating nucleophile.

38. 35. The compound of claim 34, wherein the lipid comprises two hydrophobic moieties.

39. 35. The compound of claim 34, wherein the lipid comprises a phospholipid, a glycerolipid, a dialkylamine, or a combination thereof.

40. 35. The compound of claim 34, wherein the lipid comprises 1,2-dimyristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or a combination thereof.

41. 35. The compound of claim 34, having a hydrolysis rate determined at least in part by L.

42. 35. The compound of claim 34, having a half-life for hydrolysis in 50% human plasma of about 10 minutes or less.

43. 35. The compound of claim 34, having a half-life for hydrolysis in 50% human plasma of about 120 hours or more.

44. A composition comprising a compound according to claim 1, 14, 25 or 34.

45. 45. The composition of claim 44, further comprising a cationic or ionizable lipid.

46. 46. ​​A method of eliciting a protective immune response in an animal, comprising administering to the animal an effective amount of the composition of claim 45.

47. 47. The method of claim 46, wherein the administering step comprises delivering the composition to the animal via a subcutaneous, intravenous, intramuscular, intradermal or aerosol route.

48. 46. ​​A method of treating a disorder or disease in an animal, comprising administering to the animal an effective amount of the composition of claim 45.

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