Lipid-polymer conjugates and lipid nanoparticles incorporating the same

Abstract

Lipid-polymer conjugates and associated lipid nanoparticles are described herein. Such conjugates can comprise polyoxazolines and / or poly oxazines, thereby obviating the use of PEG-based treatments.

Description

[0001] LIPID-POLYMER CONJUGATES AND LIPID NANOPARTICLES INCORPORATING THE SAME

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority pursuant to Article 8 of the Patent Cooperation Treaty to United States Provisional Patent Application Serial Number 63 / 693,393 filed September 11, 2024 which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present application relates to lipid-polymer conjugates and, in particular, to lipidpolymer conjugates comprising polyoxazolines and / or polyoxazines, and incorporation of such conjugates into lipid nanoparticles.

[0006] BACKGROUND

[0007] Nucleic acid delivery systems face challenging barriers such as off-target immune responses and low delivery efficiency. Genetic material, such as plasmid DNA (pDNA), messenger RNA (mRNA), and silencing RNA (siRNA) amongst others, is challenging to deliver in vivo due to degradation by DNases and RNAases, inefficient delivery into the cell, and lysosomal entrapment and degradation. Polymer-mediated gene delivery, for example, focuses on combining cationic polymers with negatively charged genetic material to form polyion complexes (polyplexes). One of the very well-studied cationic polymers used for plasmid delivery is a block copolymer of polyethyleneimine (PEI) and polyethylene glycol (PEG). Though PEI-PEG based polyplexes have a high transfection efficiency, high molecular weight of PEI is required for efficient transfection, which makes these polyplexes cytotoxic and unsuitable for in vivo application. The addition of PEG results in a beneficial “stealth” effect allowing for enhanced circulation in vivo. Due to its relative inertness and “stealth” property, PEG quickly became used in many cancer treatments, such as in breast and ovarian cancer drugs. PEG is now used as the primary polymer-lipid component in lipid nanoparticles (LNPs), which have become the preferred delivery vehicle for nucleic acids. However, the ubiquity of PEG is problematic in causing the rise of anti-PEG-antibodies, which decreases the efficacy of life-saving PEG-based treatments. A recent study reports that 72% of individuals have detectable levels of PEG antibodies, which is driving a significant need for alternative delivery compositions and systems. SUMMARY

[0008] In view of the foregoing, lipid-polymer conjugates and associated lipid nanoparticles are described herein. Such conjugates can comprise polyoxazolines and / or polyoxazines, thereby obviating the use of PEG-based treatments. In some embodiments, a conjugate comprises a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is of formula (I): wherein R1is selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2, and n is 5 to 500.

[0009] In another aspect, a conjugate comprises a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is formed of monomer selected from the group consisting of 2- methyl-2-oxazoline, 2-methyl-2oxazine, and 2-ethyl-2-oxazine.

[0010] In another aspect, a conjugate comprises a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II): wherein R1is selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, alkenyl, alkynyl, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10. The copolymer, in some embodiments, has a random or gradient architecture of monomer of the hydrophilic and functional components. Alternatively, the copolymer can be a block copolymer wherein the hydrophilic component and functional component constitute separate blocks.

[0011] In some embodiments of conjugates described herein, the functional component of the copolymer is located distal to the linker and lipid. For example, one or more blocks of monomer of formula (II) can be positioned distal to the linker and lipid. Similarly, in embodiments wherein the copolymer has a gradient architecture, the majority of monomer of formula (II) can be positioned distal to the linker and lipid. Moreover, in other embodiments, monomer of formula (II) can be positioned proximate the linker and lipid. In such embodiments, monomer of formula (II) can be in block form, or exhibit a gradient distribution.

[0012] In another aspect, a conjugate comprises a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises differing monomeric species of the formula: wherein R1of each of the differing monomeric species is independently selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2. In some embodiments, for example, R1of a monomer forming the copolymer is -C(O)OR2. The copolymer can exhibit a random architecture or block architecture, in some embodiments. For example, one or more of the differing monomeric species can be present as a block. In other embodiments, the copolymer can exhibit a gradient architecture. In such embodiments, the majority of one monomeric species can be positioned distal to the linker and lipid. Alternatively, the majority of one monomeric species can be positioned proximate to the linker and lipid.

[0013] In another aspect, a conjugate comprises a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises differing monomeric species of the formula: wherein R1of each of the differing monomeric species is independently selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, alkenyl, alkynyl, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10. The copolymer can exhibit a random architecture or block architecture, in some embodiments. For example, one or more of the differing monomeric species can be present as a block. In other embodiments, the copolymer can exhibit a gradient architecture. In such embodiments, the majority of one monomeric species can be positioned distal to the linker and lipid. Alternatively, the majority of one monomeric species can be positioned proximate to the linker and lipid.

[0014] In another aspect, lipid nanoparticles are described herein. In some embodiments, a lipid nanoparticle comprises a conjugate including a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is described above, including homopolymer of formula (I) above. In another aspect, a lipid nanoparticle comprises a conjugate including a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II) above. The copolymer, in some embodiments, has a random or gradient architecture of monomer of the hydrophilic and functional components. Alternatively, the copolymer can be a block copolymer wherein the hydrophilic component and functional component constitute separate blocks.

[0015] Lipid nanoparticles described herein can further comprise at least one of ionizable lipids and / or helper lipids. Additionally, lipid nanoparticles described herein are employed for the delivery of various biomolecular species and / or small molecules in the cellular environment. The lipid nanoparticles, for example, can contain biomolecular species including nucleic acids, oligonucleotides, proteins, or mixtures thereof. Without limiting this invention to a specific manufacturing method, lipid nanoparticles can be produced using standard mixing techniques known in the art. For example, a solution of nucleic acids in aqueous media can be mixed with a solution of one or more different lipids in a water-miscible organic solvent (e.g., ethanol).

[0016] These and other embodiments are further described in the following detailed description.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 illustrates amidation and esterification of functional monomer according to some embodiments.

[0019] FIG. 2 illustrates various functional groups that can be incorporated into monomer of the functional component via amidation or esterification according to some embodiments.

[0020] FIGS. 3 A and 3B compare size and poly dispersity, respectively, of lipid nanoparticles according some embodiments described herein pre- and post-lyophilization relative to prior nanoparticle architectures.

[0021] FIG. 4 illustrates a series of lipophilically carbamate prodrugs of resiquimod operable to anchor the drug to the lipid nanoparticle shell according to some embodiments.

[0022] FIGS. 5-8 illustrate1HNMR, gel permeation chromatography, and FTIR spectra of PMeOx homopolymers according to some embodiments.

[0023] FIGS. 9-12 illustrate 'HNMR, gel permeation chromatography, and FTIR spectra of PMeOx homopolymers according to some embodiments. FIG. 13 illustrates homopolymer (PMeOx) synthesis using sodium azide as the terminating agent to enable subsequent click chemistry coupling with lipids comprising DBCO.

[0024] FIGS. 14A and 14B illustrate UV-VIS spectra monitored at 310 nm confirming the coupling of the homopolymers of Table 2 with the DBCO-PE phopsholipids.

[0025] FIG. 15 provides a table detailing lipid nanoparticle characterization according to some embodiments.

[0026] FIGS 16A-16D quantify average transfection efficiency ofLNP formulations descripbed herein relative to individual cell lines, according to some embodiments.

[0027] FIG. 16E illustrates variance of lipid nanoparticle transfection across differing cell types according to some embodiments.

[0028] FIG. 16F illustrates the ratio of DC 2.4, 4T1, and IC21 transfection levels relative to HEK293 expression with various lipid nanoparticle compositions according to some embodiments.

[0029] FIG. 17A illustrates in vivo imaging of a side view of mice after injection of Luciferase mRNA loaded lipid nanoparticles, according to some embodiments.

[0030] FIG. 17B illustrates in vivo imaging of a ventral view of mice after injection of Luciferase mRNA loaded lipid nanoparticles, according to some embodiments.

[0031] FIGS. 18A-18B illustrate muscle expression in female and male mice, respectively, at 4 and 24 hours post injection of mRNA loaded nanoparticles according to some embodiments.

[0032] FIGS. 18C-18D illustrate corresponding liver expression data for the female and male mice, respectively.

[0033] FIG. 18E shows that muscle area under the curve (AUC) for lipid nanoparticles having composition and architecture described herein was significantly higher relative to PEG liquid nanoparticles.

[0034] FIG. 18F illustrates reduced liver accumulation in female mice of lipid nanoparticles having composition and architecture described herein.

[0035] FIG. 18G provides a ratio of muscle gene expression at 24 hours relative to 4 hours.

[0036] FIG. 18H provides a ratio of muscle gene expression at 24 hours relative to 4 hours.

[0037] FIG. 181 provides a ratio of liver AUC to muscle AUC in female and male mice.

[0038] FIG. 18J provides a J-score for lipid nanoparticle compositions according to some embodiments. FIG. 19A illustrates normalizes ex vivo luminescence (RLU / g tissue) in muscle tissue excised from mice as a function of LNP formulation according to some embodiments.

[0039] FIG. 19B illustrates normalizes ex vivo luminescence (RLU / g tissue) in liver tissue excised from mice as a function of LNP formulation according to some embodiments.

[0040] FIG. 19C illustrates normalizes ex vivo luminescence (RLU / g tissue) in kidney tissue excised from mice as a function of LNP formulation according to some embodiments.

[0041] FIG. 19D illustrates normalizes ex vivo luminescence (RLU / g tissue) in spleen tissue excised from mice as a function of LNP formulation according to some embodiments.

[0042] FIG. 19E illustrates normalizes ex vivo luminescence (RLU / g tissue) in lymph node tissue excised from mice as a function of LNP formulation according to some embodiments.

[0043] FIG. 19F illustrates normalizes ex vivo luminescence (RLU / g tissue) in lung tissue excised from mice as a function of LNP formulation according to some embodiments.

[0044] FIG. 19G illustrates normalizes ex vivo luminescence (RLU / g tissue) in heart tissue excised from mice as a function of LNP formulation according to some embodiments.

[0045] FIG. 19H illustrates normalizes ex vivo luminescence (RLU / g tissue) in brain tissue excised from mice as a function of LNP formulation according to some embodiments.

[0046] FIG. 20A illustrates the results of in vitro transfection of LNP formulations containing EtOx-20 LNPs frozen with and without different cryoprotectants compared to fresh (never frozen) formulations.

[0047] FIG. 20B illustrates the results of in vitro transfection MeOx-DPIO and DP20 formulations with glycerol (5%) and / or sucrose (9%) cryoprotection compared to standard PEG formulations.

[0048] FIG. 21 illustrates deprotection of MestOx polymer.

[0049] FIG. 22 illustrates amidation of MestOx polymer, according to some embodiments.

[0050] FIG. 23 illustrates amidation of MestOx polymer, according to some embodiments.

[0051] FIG. 24 illustrates azide terminated methyl oxazine polymer synthesized according to methods described herein.

[0052] FIG. 25 illustrates polymer-lipid coupling in the formation of conjugates described herein according to some embodiments.

[0053] FIG. 26 provides the results of the transfection study employing lipid nanoparticles having composition and architecture described herein according to some embodiments. FIG. 27 illustrates ex vivo luminescence in organs excised from mice injected with nanoparticle formulation described herein according to some embodiments.

[0054] DETAILED DESCRIPTION

[0055] Embodiments described herein can be understood more readily by reference to the following detailed description and examples and their previous and following descriptions. Elements, apparatus and methods described herein, however, are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

[0056] I. Lipid-Polymer Conjugates

[0057] Lipid-polymer conjugates and associated lipid nanoparticles are described herein. In some embodiments, a conjugate comprises a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is of formula (I): wherein R1is selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2, and n is 5 to 500. In some embodiments, R1is -C(O)OR2. For example, monomer of the homopolymer of formula (I) can be selected from the group consisting of 2-methoxycarboxyethyl-2-oxazoline, 2-carboxyethyl-2- oxazoline, 2-methoxycarboxyethyl-2-oxazine, and 2-carboxyethyl-2-oxazine. In some embodiments, R1is -C(O)NR3R4. FIG. 2 provides various examples of NR3R4moieties for inclusion in the homopolymer. In some embodiments, R1of the monomer is initially incorporated as a carboxyl moiety that is subsequently amidated, as illustrated in FIG. 1.

[0058] In another aspect, a conjugate comprises a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is formed of monomer selected from the group consisting of 2- methyl-2-oxazoline, 2-hydroxymethyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-methyl-2oxazine, 2- ethyl-2-oxazine, and 2-hydroxymethyl-2-oxazine.

[0059] In another aspect, a conjugate comprises a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises differing monomeric species of the formula: wherein R1of each of the differing monomeric species is independently selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NCh, and wherein m is 1 or 2. In some embodiments, for example, R1of a monomer forming the copolymer is -C(O)OR2. In some embodiments, the differing monomeric species are selected form the group consisting of 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-hydroxymethyl-2- oxazoline, 2-propyl-2-oxazoline, 2-butyl-2-oxazoline, 2-pentyl-2-oxazoline, 2-methyl-2-oxazine, 2-ethyl-2-oxazine, 2-hydroxymethyl-2-oxazine, 2-propyl-2-oxazine, 2-butyl-2- oxazine, and 2- pentyl-2-oxazine. The copolymer can exhibit a random architecture or block architecture, in some embodiments. For example, one or more of the differing monomeric species can be present as a block. In other embodiments, the copolymer can exhibit a gradient architecture. In such embodiments, the majority of one monomeric species can be positioned distal to the linker and lipid. Alternatively, the majority of one monomeric species can be positioned proximate to the linker and lipid.

[0060] In another aspect, a conjugate comprises a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises differing monomeric species of the formula: wherein R1of each of the differing monomeric species is independently selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, alkenyl, alkynyl, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10. The copolymer can exhibit a random architecture or block architecture, in some embodiments. For example, one or more of the differing monomeric species can be present as a block. In other embodiments, the copolymer can exhibit a gradient architecture. In such embodiments, the majority of one monomeric species can be positioned distal to the linker and lipid. Alternatively, the majority of one monomeric species can be positioned proximate to the linker and lipid.

[0061] In another aspect, a conjugate comprises a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II):

[0062] wherein R1is selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, alkenyl, alkynyl, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10.

[0063] Turning now to specific components, the hydrophilic component can comprise non-ionic oxazoline monomer or non-ionic oxazine monomer. In some embodiments, the oxazoline monomer is selected from the group consisting of 2-methyl-2-oxazoline, 2-hydroxymethyl-2- oxazoline, 2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2-isopropyl-2-oxazoline, 2-butyl-2- oxazoline, 2-pentyl-2-oxazoline, and combinations thereof. In some embodiments, the oxazine monomer is selected from the group consisting of 2-methyl-2-oxazine, 2-hydroxymethyl- oxazine, 2-ethyl-2-oxazine, 2-propyl-2-oxazine, 2-isopropyl-2-oxazine, 2-butyl-2-oxazine, 2- pentyl-2-oxazine, and combinations thereof. The hydrophilic component can be formed of a single monomeric species or a mixture or combination of monomeric species. For example, the hydrophilic component can be formed of two or more of 2-methyl-2-oxazoline, 2-hydroxy-2- oxazoline, 2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2-isopropyl-2-oxazoline, and 2-butyl-2- oxazoline, in some embodiments. The hydrophilic component, in some embodiments, is formed of two or more of 2-methyl-2-oxazine, 2-hydroxy-2-oxazine, 2-ethyl-2-oxazine, 2-propyl-2- oxazine, 2-isopropyl-2-oxazine, and 2-butyl-2-oxazine.

[0064] As described above, monomer of the functional component is of formula (II) above. In some embodiments of formula (II), R1is -C(O)OR2. For example, in some embodiments, monomer of formula (II) can be selected from the group consisting of 2-methoxycarboxyethyl-2- oxazoline, 2-carboxyethyl-2-oxazoline, 2-methoxycarboxyethyl-2-oxazine, 2-carboxyethyl-2- oxazine, and mixtures thereof. In some embodiments, R1is -C(O)NR3R4. FIG. 2 provides various examples of NR3R4moieties for inclusion in the functional component of the copolymer. In some embodiments, R1of the monomer is initially incorporated as a carboxyl moiety that is subsequently amidated or esterified, as illustrated in FIG. 1. In this way, various functional groups or moieties can be incorporated into monomer of the functional component. In some embodiments, two or more differing functional groups can be incorporated into the functional component via monomer amidation or esterification.

[0065] The functional component can employ a single monomeric species of formula (II) or can employ various mixtures of monomeric species contemplated by formula (II). In some embodiments, the functional component comprises 1 to 15 monomeric units of Formula (II). In other embodiments, monomer of Formula (II) accounts for 10 percent to 95 percent of the copolymer.

[0066] The copolymer, in some embodiments, has a random or gradient architecture of monomer of the hydrophilic and functional components. Alternatively, the copolymer can be a block copolymer wherein the hydrophilic component and functional component constitute separate blocks.

[0067] In some embodiments of conjugates described herein, the functional component of the copolymer is located distal to the linker and lipid. For example, one or more blocks of monomer of formula (II) can be positioned distal to the linker and lipid. In some embodiments, the conjugate is of the formula: wherein R1, m and p are defined in formula (II) above, G is selected from the group consisting of alkyl and cycloalkyl, each optionally substituted with hydroxyl, -SH, and C(O)OR5, wherein R5is selected from the group consisting of hydrogen and alkyl, L is a linker, E is an end of the copolymer, n is 1 or 2, and x and y are independently 1 to 500. In some embodiments, E can be a targeting ligand or moiety, as described below. In other embodiments, the functional block is proximate the linker (L) and lipid.

[0068] In embodiments wherein the copolymer has a gradient architecture, the majority of monomer of formula (II) can be positioned distal to the linker and lipid. Moreover, in other gradient embodiments, the majority of monomer of formula (II) can be positioned proximate the linker and lipid.

[0069] Conjugates described herein comprising homopolymer or copolymer also include a lipid coupled to the homopolymer or copolymer via a linker, L. Any desired lipid consistent with the technical objectives described herein can be employed. In some embodiments, the lipid is a phospholipid, a cationic lipid, ionizable lipid, a steroid compound such as cholesterol, or mixtures thereof. The lipid can comprise a single hydrophobic tail or multiple hydrophobic tails. Hydrophobic tails of the lipid can be the same or different. In some embodiments, for example, hydrophobic tails exhibit differing compositions, branching, and / or chain lengths. The lipid, for example, can be selected from the group consisting of distearoylphosphoethanolamine (DSPE), diacylglyceral (DAG), dimyristoyl glycerol (DMG), cholestryl, and tocopheryl. In some embodiments, the lipid is phosphatidyl glycerol (PG), phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidic acid (PA), phosphatidyl inositol (Pl), phosphatidyl serine (PS), or combinations thereof.

[0070] The linker, L, can comprise a reaction product of click-chemistry moieties of the polymer and lipid. Suitable click chemistries for joining the homopolymer or copolymer and lipid include BCN, DBCO, TCO, tetrazine, alkyne, and azide. Additional coupling methods, such as NHS- Ester or maleimide chemistry, are applicable to produce a linker. Alternatively, the linker can be derived from a process whereby polymerization of the homopolymer or copolymer is conducted starting from the lipid. The linker can also be derived from a process whereby polymerization of homopolymer or copolymer is terminated by the linker and / or the lipid. The linker can be degradable via hydrolysis or enzymatic processes, such as carbamate, ester, peptide, and polypeptide.

[0071] Further, in some embodiments, the conjugate can also comprise a reactive moiety for attaching a targeting ligand at an end of the copolymer distal to the lipid. In some embodiments, the targeting ligand is selected from the group consisting of an aptamer, antibody, antibody fragment, peptide, and small molecule. In other embodiments, the targeting ligand is coupled to the carboxy-functionalized alkyl-oxazoline monomer, carboxy-functionalized-oxazoline monomer, carboxy-functionalized alkyl-oxazine monomer, or carboxy-functionalized-oxazine monomer of the copolymer. In some embodiments, the targeting ligand is coupled to an alkene or alkyne functionalize monomer of the copolymer.

[0072] II. Lipid Nanoparticles

[0073] In another aspect, lipid nanoparticles are described herein. In some embodiments, a lipid nanoparticle comprises a conjugate including a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is of formula (I) above.

[0074] In another aspect, a lipid nanoparticle comprises a conjugate including a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II) above. The copolymer, in some embodiments, has a random or gradient architecture of monomer of the hydrophilic and functional components. Alternatively, the copolymer can be a block copolymer wherein the hydrophilic component and functional component constitute separate blocks. Conjugates of lipid nanoparticles described herein can have any composition and / or properties detailed in Section I above.

[0075] Lipid nanoparticles can comprise any amount of conjugate consistent with the technical objectives described herein. In some embodiments, a conjugate is present in a lipid nanoparticle in an amount of 0.01-10 mol.% or 0.5-5 mol.% of the lipids comprising the nanoparticle.

[0076] Lipid nanoparticles described herein can further comprise at least one of ionizable lipids and helper lipids. In some embodiments, helper lipids include structural ligands, a steroid compound such as cholesterol, or mixtures thereof.

[0077] Additionally, lipid nanoparticles described herein are employed for the delivery of various biomolecular species in the cellular environment. The lipid nanoparticles, for example, can contain biomolecular species including nucleic acids, oligonucleotides, proteins, or mixtures thereof. The lipid nanoparticles can also contain small molecule organic compounds, including but not limited to physiologically active compounds, such as chemotherapeutic drugs, immunomodulators, inhibitors, as well as other compounds. Nucleic acids include DNA, such as single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA), plasmid DNA (p-DNA), and mixtures thereof. Nucleic acids additionally comprise RNA, including messenger RNA (mRNA), transfer RNA (RNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), circular RNA (circRNA), microRNA (miRNA), small-activating RNA (sa-RNA), long noncoding RNAs (IncRNAs), and mixtures thereof. Nucleic acids also comprise mixtures of RNA and DNA. The nucleic acids may be naturally occurring or synthetic. Nucleic acids, in some embodiments, comprise one or more modifications. For example, synthetic mRNA can comprise one or more uridine analogs, such as pseudouridine (\| / ) and / or N1-methyl -pseudourine (ml\| / ). Additionally, modifications of nucleic acids and / or oligonucleotides described herein also include protein modifications. The lipid nanoparticles can also contain proteins and polypeptides including but not limited to those that can bind to the nucleic acid. In some embodiments, a nucleic acid or oligonucleotide is modified with one or more proteins, including enzymes such as nucleases. For example, a nucleic acid of a polyion complex described herein can be Cas9 gRNA.

[0078] In some embodiments, small molecules carried by lipid nanoparticles described herein are biologically active and, in some embodiments, can interact with immune receptors and affect immune response in the body. Small molecules carried by the lipid nanoparticles, for example, can include Toll-like receptor 4 (TLR4) agonists, such as monophosphoryl lipid A (MPLA), Toll-like receptor 7 and 8 (TLR7 / 8) agonist such as: Resiquimod (R848), or Imiquimod; or TLR4 agonist CSF1R inhibitor such as Pexidartinib (PLX3397), (R)-limonene 6-monooxygenase (PLX5622), and Sotuletinib (BLZ945), or PI3K5 Inhibitor such as Idelalisib (GS1101), a pl 10 inhibitor.

[0079] Small molecules carried by lipid nanoparticles described herein, in some embodiments, are coupled to lipid or lipophilic tails via cleavable or non-cleavable linkers. The resulting modified small molecules are mixed with lipid nanoparticles described herein, wherein the lipid or lipophilic tails are operable to engage with lipids of the lipid nanoparticles. In some embodiments, the lipid or lipophilic tails insert into the shell of the lipid nanoparticles. Alternatively, the modified small molecules can be mixed with conjugates described herein alone with free lipids during formation of the lipid nanoparticles. In some embodiments, the lipid or lipophilic tails insert into the shell of the lipid nanoparticles.

[0080] In some embodiments, the lipophilic tail comprises one or more aliphatic chains or moieties. Suitable aliphatic chains, in some embodiments, are CL - C30, such as C10 - C20. Length of the aliphatic chain can be varied as desired to the extent the aliphatic tail anchors the small molecule to the lipid nanoparticles via insertion or interaction with the lipid nanoparticle. Suitable aliphatic chains can be linear or branched and optionally contain one or more points of unsaturation. In some embodiments, the lipophilic tail comprises two or more aliphatic chains. Additionally, in some embodiments, the lipophilic tail can comprise one or more cyclic, heterocyclic, aryl or heteroaryl moieties. Such moieties can comprise plurality of fused or unfused ring structures. In some embodiments the lipophilic tail comprises sterols, such as cholesterol or derivatives of cholesterol.

[0081] The small molecule can be covalently bound to the lipid or lipophilic tail. In some embodiments, the small molecule can be bound to the lipid or lipophilic tail via a moiety selected from the group consisting of carbamate, ester, amide, disulfide, imine, enamine, beta amino ester, malemeide, and diazo. Specific identity of the moiety binding the small molecule to the lipid or lipophilic tail can be dependent on several considerations including, but not limited, to specific chemical identity of the small molecule and the local biological environment in which the composite exosome will be disposed. For example, the moiety binding the small molecule to the tail can be cleaved by one or more species or conditions in the local environment. In some embodiments, the moiety is cleaved by an enzyme or other protein species in the local biological environment. Alternatively, the moiety can be cleaved by pH conditions (acidic or basic) of the local biological environment. The local biological environment can be within a cell or in the intercellular environment.

[0082] Cleaving the moiety releases the small molecule into the local biological environment. Coupling the small molecule to the lipid or lipophilic tail and subsequent cleavage of the small molecule does not alter or substantially alter the biological activity and / or structure of the small molecule. Further, release kinetics of the small molecule into the local biological environment, in some embodiments, can be tailored via selection of the tail identity. In some embodiments, shorter the lipid or lipophilic tail structures, such as shorter aliphatic chains, can demonstrate a faster release profile of the small molecule. The shorter aliphatic chains, for example, do not anchor as deeply in the lipid nanoparticle, thereby facilitating accelerated release kinetics relative to longer aliphatic chains. FIG. 4 illustrates a series of lipophilically carbamate prodrugs of resiquimod operable to anchor the drug to the lipid nanoparticle shell according to some embodiments. Small molecules modified with lipid or lipophilic tails are can be operable for use with lipid nanoparticles of any desired construction, including lipid nanoparticles described herein. Therefore, small molecules modified with lipid or lipophilic tails are not limited to inclusion only in lipid nanoparticles described herein, but can insert into lipid membranes of various lipid nanoparticles.

[0083] In some embodiments, lipid nanoparticles having construction and properties described herein exhibit an encapsulation efficiency for a biomolecular species or small molecule of at least 70 percent or at least 80 percent. Encapsulation efficiency of the lipid nanoparticles can range from 70-99 percent, 80-99 percent, or 90-99 percent, in some embodiments. Moreover, lipid nanoparticles having construction and properties described herein can exhibit a poly dispersity index (PDI) of 0.05 to 0.25 or 0.05 to 0.2, in some embodiments. As set for the in the example herein, PDI is measured by dynamic light scattering (DLS). Lipid nanoparticles having construction and properties described herein can also exhibit negative zeta potential. In some embodiments, the lipid nanoparticles have a zeta potential of -2 mV to -30 mV, -2.5 mV to -20 mV, or -2 mV to - 10 mV.

[0084] Lipid nanoparticles having construction and properties described herein can be lyophilized and subsequently reconstituted. FIGS. 3A and 3B compare size and polydispersity, respectively, of lipid nanoparticles according some embodiments described herein pre- and postlyophilization relative to prior nanoparticle architectures. Following lyophilization, all four formulations increased in both size and PDI following lyophilization. Interestingly, the PMeOx LNPs showed the smallest increase in size (28.4%), followed by “Moderna-like” LNPs (32.7%), PEtOx LNPs (40%), and 16:0 PE-PEG LNPs (47.6%). In addition, PMeOx LNPs displayed the lowest average PDI (0.21) of the four formulations after lyophilization. These preliminary results show potential for POx polymers to provide better stability for lyophilized LNPs than existing formulations.

[0085] In addition, it was discovered that encapsulation efficiency reduced dramatically for all LNPs following lyophilization. Both the PMeOx LNPs and “Moderna-like” LNPs showed the smallest reductions in encapsulation efficiency (-40%), while the PEtOx LNPs and 16:0 PE- PEG LNPs showed greater reductions (-50%). The PMeOx and “Moderna-like” LNPs also retained the highest encapsulation efficiencies following lyophilization, although it is noted that their initial encapsulation efficiencies were higher than the PEtOx and 16:0 PE-PEG LNPs. This data further implies that PMeOxylated lipids may provide a suitable alternative to PEGylated lipids in existing formulations. Encapsulation efficiencies are provided in Table 1. Table 1

[0086] In another aspect, methods of making lipid nanoparticles are described herein. In some embodiments, a method of making a lipid nanoparticle comprises providing a mixture including free lipids and a conjugate comprising a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is of formula (I): wherein R1is selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2, and n is 5 to 500. The free lipids and the conjugate are formed into a shell of the lipid nanoparticle, wherein the homopolymer resides on an exterior of the shell.

[0087] In another aspect, a method comprises providing a mixture including free lipids and a conjugate comprising a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II):

[0088] wherein R1is selected from the group consisting of consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10. The free lipids and the conjugate are formed into a shell of the lipid nanoparticle, wherein the copolymer resides on an exterior of the shell.

[0089] In another aspect, a method comprises providing a mixture including free lipids and a conjugate comprising a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises differing monomeric species of the formula: wherein R1of each of the differing monomeric species is independently selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2. The free lipids and the conjugate are formed into a shell of the lipid nanoparticle, wherein the copolymer resides on an exterior of the shell.

[0090] In another aspect, a method comprises providing a mixture including free lipids and a conjugate comprising a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises differing monomeric species of the formula: wherein R1of each of the differing monomeric species is independently selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10. The free lipids and the conjugate are formed into a shell of the lipid nanoparticle, wherein the copolymer resides on an exterior of the shell.

[0091] In some embodiments, of methods described herein, the mixture further comprises one or more small molecules and / or biomolecular species, wherein the small molecules and / or biomolecular species are incorporated into the nanoparticle interior. The biomolecular species can have any identity described herein, including nucleic acids and proteins. Small molecules can comprise pharmaceuticals intended to treat various indications. Moreover, conjugates employed in methods described herein can have any composition and / or properties detailed in Section I above, including various copolymer architectures such as random, block, or gradient architecture.

[0092] Additional embodiments of compositions and methods described herein are further provided in the following Examples. EXAMPLE 1 - Homopolymer Synthesis

[0093] Homopolymers having the properties discloses in Table 2 were synthesized via living cationic ring opening polymerization of 2-methyl-2-oxazoline and 2-ethyl-2-oxazoline monomer to produce homopolymers PMeOx and PEtOx, respectively. Table 2 provides characterization data for the synthesized homopolymers.

[0094] Moreover, the preparation of random copolymers, gradient copolymers and block copolymers, is described in detail, e.g., by R. Luxenhofer and R. Jordan, Macromolecules 39, 3509-3516 (2006), T. Bonne et al., Colloid. Polym. Sci , 282, 833-843 (2004) or T. Bonne et al. Macromol. Chem. Phys. 2008, 1402-1408, (2007).

[0095] Table 2 - Characterization of polymers synthesized for this study centered around the molecular weight and degree of polymerization of PEG-2000. Individual polymer batches were synthesized.

[0096] Polymer Batch Code Nominal DP HNMR DP HNMR MnGPC M„ GPC MwPDI

[0097] V6 10 11 992 673 678 1.007

[0098] V4 20 18 1587 1004 1083 1.039

[0099] PMeOx

[0100] V5 40 56 4817 2832 3929 1.167

[0101] V7 80 85 7282 4976 7633 1.275

[0102] V3 10 11 1146 972 995 1.024

[0103] VI 20 24 2433 1887 2003 1.061

[0104] PEtOx

[0105] V2 40 57 5700 5686 6125 1.077

[0106] V4 80 96 9561 13562 14609 1.077 FIGS. 5-8 illustrate 'HNMR, gel permeation chromatography, and FTIR spectra of PMeOx of Table 2. FIGS. 9-12 illustrate ’HNMR, gel permeation chromatography, and FTIR spectra of PMeOx of Table 2.

[0107] EXAMPLE 2 - Conjugate Synthesis Homopolymers and copolymers of conjugates described herein are prepared by polymerization methods known in the art, as illustrated in Example 1. FIG. 13 illustrates homopolymer (PMeOx) synthesis using sodium azide as the terminating agent to enable subsequent click chemistry coupling with lipids comprising DBCO. POx polymers terminated with azide groups of Table 2 were incubated with DBCO-PE phopsholipids in a 3: 1 molar ratio in ethanol resulting in copper-free click chemistry coupling between the azide and DBCO groups. The coupling reaction was monitored via the absorbance of free DBCO groups at 310 nm, wherein decrease in the DBCO absorbance evidences the coupling of lipid and homopolymer. FIGS. 14A and 14B illustrate UV-VIS spectra monitored at 310 nm confirming the coupling of the homopolymers of Table 2 with the DBCO-PE phopsholipids.

[0108] EXAMPLE 3 - Lipid Nanoparticle Synthesis

[0109] Conjugates synthesized according to Example 2 were formed into lipid nanoparticles according to the following procedure. In the present example, the lipid nanoparticles were formulated with four lipid components: a cationic ionizable lipid, cholesterol, an additional helper lipid, and the polymer-lipid conjugates of Example 2. The lipid nanoparticles of the present example employed SM-102 ionizable lipid and DSCP as the additional helper lipid. The four lipids can be combined in any desired molar ratio consistent with the technical objectives herein. For the present example, the combination ratio was 50:38.5: 10:1.5. The quantity of lipids was determined using a 19.35:lwt / wt ratio for lipids:mRNA. To produce the lipid nanoparticles, the lipids were solvated in ethanol at about 8mM concentration, while the corresponding amount of mRNA was dissolved in 10 mM acetate buffer (7.7 mM acetic acid, 2.3 mM sodium acetate, pH 4.15). The lipids and mRNA were combined using a pump-driven impinged jet mixer (Nova Benchtop impinged jet mixed from Helix Biotechjat a 3: 1 organic:aqueous flow rate ratio and a predetermined total flow rate of 8 mL / min. The lipid nanoparticles were then collected for purification.

[0110] The collected lipid nanoparticles encapsulating the mRNA and placed in dialysis to remove excess ethanol from the formulation. Dialysis tubing with a molecular weight cutoff of 12-14 kDa is used to dialyze LNPs against a lOmM acetate buffer, pH 4.15, for at least 8 hours at room temperature. LNPs were then transferred to dialyze against lx PBS at physiological pH for at least 8 more hours at room temperature. LNPs were then recovered, and if necessary, concentrated using 100 kDa ultra centrifugal filtration tubes. To facilitate long-term storage, 10% weight / volume sucrose was added to the dialyzed, concentrated LNP solution. Finally, the LNPs are filtered using a polyethersulfone (PES) membrane with 0.4 / rm pore diameters.

[0111] Lipid nanoparticles with 1.5 mol % DMG-PEG2000 were synthesized as a comparative. Characterization results of the lipid nanoparticles formed with the PMeOx and PEtOx conjugates of Example 2 are provided in the Table 3 of FIG. 15. Dynamic light scattering (DLS) was used to evaluate the size and poly dispersity index of the lipid nanoparticles. Nanoparticle tracking analysis was also employed in characterizing the lipid nanoparticles, including determination of zeta potential of the particles. A Ribogreen assay was used to evaluate the mRNA encapsulation efficiency of the different LNP solutions. Briefly, LNPs were diluted 200x and plated in 200 pL wells, where they were incubated for 5 minutes with Ribogreen dye, which binds to free mRNA and fluoresces linearly with concentration but is membrane impermeable. Some LNPs were lysed with 0.5% v / v Triton X-100, which exposed all encapsulated and unencapsulated mRNA to the Ribogreen dye. The encapsulation efficiency was calculated from the fluorescent intensities using Equation 1 below. Four wells per test condition were averaged to calculate encapsulation efficiency using Equation 1.

[0112] EXAMPLE 4 - In vitro Transfection Study

[0113] In vitro transfection of multiple cell lines with lipid nanoparticles of Example 3 was conducted. To assess relative expression levels and potential cell-type tropism, we evaluated the in vitro transfection efficiency of our POx- and PEG-based LNPs across three cell lines: HEK293, DC2.4 (a murine dendritic cell line), and 4T1 (a murine triple-negative breast cancer cell line). Each individual batch was independently tested in these cell lines to evaluate any batch-to-batch variation in transfection efficiency.

[0114] HEK293, DC2.4, or 4T1 cells were seeded in appropriate medium at 30,000 cells / well in a 96-well plate with n=6 wells prepared for each treatment group including blank and mRNA alone controls. HEK293 was cultured in DMEM with 10% FBS and 1% antibiotic / antimycotic. DC2.4 were cultured in MEM media with 10% FBS, 1% antibiotic / antimycotic, IX L-glutamine reagent, and 0.0054X 2-mercaptoethanol. 4T1 cells were cultured in RPMI media with 10% FBS and 1% antibiotic / antimycotic. Cells were allowed to adhere overnight. LNPs were diluted in appropriate cell culture media to 1 pg / mL total mRNA. For treatment, media was removed and 100 pL of LNPs were added to each well (100 ng mRNA / well). Cells were incubated for 24 hours. At 24 hours, media was removed and cells were lysed in 30 pL of IX Triton and incubated with shaking at 4 °C for 1 hour. After one hour, 2 pL of each well was pipetted into a microcentrifuge tube (transparent). Sequentially and read one at a time, 50 pL of Luciferin assay reagent was added to each tube and luminescence read immediately on a ProMega GLOMAX 20 / 20 Luminometer. After luminescence readings, cell lysate was diluted 10X in 100-fold diluted Triton X-100. Cell lysates were then assayed according to standard BCA assay protocol for protein concentration while diluting standards in IX Triton. Luminescence was normalized to the pg of protein in the lysate. Each individually prepared LNP batch was assayed in vitro.

[0115] FIGS 16A-16D show the average transfection efficiency of the batches for each LNP formulation relative to the individual cell lines. Across all three cell types, the DP 10 PMeOx and DP 10 PEtOx formulated LNPs show consistently high transfection efficiency, comparable or superior to PEG-2000 LNPs in each cell line. The DP20 polymers generally show strong transfection as well but yielded statistically lower transfection than their DP 10 counterparts in all three cell lines. As the DP of the polymers increases to 40 and beyond, transfection efficiency declines sharply. Across the tested cell lines and selected polymers, we observe a consistent trend of decreasing transfection levels from HEK293 to DC2.4 to 4T1, suggesting cell-type- dependent differences in uptake or expression efficiency.

[0116] Within each LNP formulation type, all cell types very different levels of transfection, as provided in FIG. 16E. To further investigate potential differences in cell-type tropism, transfection profiles were compared across formulations, aiming to identify any preferential targeting or expression patterns among HEK293, DC2.4, 4T1, and IC21 cells. All LNP formulations were relatively poor at transfecting 4T1 an IC21 cells compared to HEK293 and DC2.4 cells (luminescence ~1% or less of HEK293 in many groups). Using HEK293 as a baseline expression level, where no formulation is expected to have inherent preferential tropism, the ratio of DC2.4, 4T1, and IC21 transfection was compared to HEK293 (FIG. 16F). It was observed that the short PMeOx DP 10 polymer exhibited increased DC2.4, 4T1, and IC21 to HEK293 transfection ratios compared to PEG-based LNPs (one-way ANOVA analysis, a=0.05). The DP 10 PEtOx polymer showed significantly improved transfection ratios as well in the 4T1 and IC21 cell lines (one-way ANOVA analysis, a=0.05). The DP20 PMeOx and PEtOx polymers exhibited not significantly different ratios in each of the cell lines. Generally, the short DP 10 polymers outperformed the DP20 polymers of the same kind. This data suggests an enhanced propensity of these short-chain POx LNPs to transfect dendritic cells, cancer cells, and macrophages relative to other formulations, like the pegylated LNPs. These improvements of short POx lipids to transfect dendritic cells highlights their possible utility and advantages over the standard PEG-LNP in a vaccine platform.

[0117] EXAMPLE 5 - In vivo Transfection Study

[0118] While in vitro and in vivo transfection efficiencies can be correlated, this is not always dispositive. All tissue types face drug delivery barriers and may transfect differently compared to the in vitro systems. To this end, the ability of lipid nanoparticles having composition and architecture described herein was evaluated for expression of mRNA in a vaccine setting. The top performing LNPs in vitro were selected for this in vivo study including DP 10 and DP20 PMeOx and PEtOx LNPs. DP40 PEtOx was also selected, and PEG-2000 LNPs were used as a clinically relevant control. Formulations for injection were prepared by mixing the two individually formulated batches into a single solution for each formulation. 325 Balb / c mice (3 female, 3 male) were injected intramuscularly in the right gluteus medius muscle with 5 pg of fLuc mRNA in a 50 pL volume (DPBS diluent). After 4 hours, mice were injected intraperitoneally with 50 pL of 30 mg / mL D-Luciferin in vivo reagent. After 5 minutes, they were put under isoflurane anesthesia, and 5 minutes later luminescence was imaged on an IVIS Spectrum in vivo Imaging System. Images were acquired of side views and ventral views ensuring no saturated pixels were present in an image. Regions of interest (ROIs) were drawn around muscle tissue (side view) and livers (ventral view) for quantification of mRNA expression. Imaging was repeated at 24 hours post-injection.

[0119] Overall, expression was high across all formulations, with PMeOx and PEtOx DP 10 having some localized hot spots of increased transfection. Additionally, from examining the images in FIG. 17A, there appears to be a greater tendency for expression to spread outside of the muscle, into tissues other than just the liver, for the DP 10 polymers compared to PEG-2000 and DP20 polymers. PEG-2000 and DP20 polymers appear to have luminescence more strictly confined to muscle and liver tissues. FIG. 17A shows the luminescence from a side-view of each mouse while FIG. 17B shows a ventral view where we could appropriately monitor full-body and liver luminescence. Notably, for the PEG-2000 based LNPs in FIG. 17B the expression in the liver increases from 4 to 24 hours post injection. Meanwhile, for each of the POx-based LNPs, the liver expression decreases from 4 to 24 hours post injection, except the short DP 10 PMeOx polymer in males, which stayed relatively stable.

[0120] Muscle expression is shown in the side view images (FIG. 17A), while liver expression is clearly viewed in the ventral view images (FIG. 17B). Statistically significant intersex differences were observed with several of the LNP formulations in both the muscle and liver at each time point (one-way ANOVA with selected comparisons between sexes within each formulation, a=0.05). Beyond statistical significance, a consistent trend emerged: in most formulations, muscle expression levels were higher in female mice compared to male mice. Male mice showed a trend of higher liver accumulation compared to females.

[0121] EXAMPLE 5 - Quantification of In Vivo Imaging

[0122] Mand liver expression levels were compared across treatment groups within each sex to evaluate how the novel POx LNPs compare to the standard PEG-2000 LNPs. FIG. 18A and FIG. 18B present muscle expression in female and male mice, respectively, at 4 and 24 hours post injection. FIG. 18C and FIG. 18D show the corresponding liver expression data. At the 4-hour time point, both PEtOx and PMeOx polymers with DP 10 show increased muscle expression in female mice while only PEtOx DP 10 showed a similar enhancement in male mice. By 24 hours, both polymers improved muscle expression in males, suggesting a time-dependent effect. In contrast, liver expression in PEG-2000 LNP-treated mice increased from 4 to 24 hours in both sexes. Notably, POx LNPs generally exhibited a decrease in liver expression over the same time period, indicating a potentially favorable biodistribution profile for vaccine applications.

[0123] FIG. 18E shows that the muscle area under the curve (AUC) for both DP 10 POx polymers was significantly higher than for PEG-2000 LNPs. Additionally, DP20 POx polymers demonstrated reduced liver accumulation in female mice (FIG. 18F). Although PMeOx-DPIO LNPs exhibited initially high liver uptake, this decreased drastically by 24 hours post-injection. To further assess biodistribution, we calculated the AUC ratio (AUC-Liver / AUC-Muscle) as a measure of each formulation’s tendency to avoid liver accumulation — a factor known to reduce vaccine efficacy and increase toxicity (FIG. 181).

[0124] While several statistically significant improvements were observed POx LNPs compared to PEG-2000 LNPs, we sought to integrate these findings into a single composite metric that captures key performance attributes: sustained expression in muscle tissue, reduced liver accumulation, and overall expression levels. To this end, a developed the “J-Score” was developed, a quantitative index that incorporates muscle durability (defined as the ratio of muscle gene expression at 24 hours to that at 4 hours; FIG. 18G) and liver accumulation (defined as the ratio of liver gene expression at 24 hours to 4 hours; FIG. 18H), as outlined in Equation 2.

[0125] Equation 2: (Muscle Durability / Liver Accumulation) * (AUC-Muscle / AUC Liver)

[0126] A higher J-Score indicates superior LNP performance, reflecting both sustained expression in the target tissue and reduced off-target accumulation. This metric enables direct comparison of formulation biodistribution by integrating multiple biodistribution and expression parameters into a single, interpretable value. As shown in FIG. 181, LNPs formulated with PEtOx-DPIO polymers achieved the highest I-Scores in both female and male mice, highlighting their strong expression profiles and reduced liver exposure — key attributes for effective and safe vaccine delivery. The J-Score can be further modified to incorporate overall expression levels for formulations with drastically different expression levels (Equation 3).

[0127] Equation 3: Log[(Muscle Durability / Liver Accumulation) * ((AUC-Muscle)2 / AUC Liver)]

[0128] EXAMPLE 6 - Ex Vivo Organ Luminescence

[0129] At 24 hours post-injection, mice were euthanized, and major organs were harvested for ex vivo luminescence analysis. To further explore trends in organ-specific expression as a function of LNP formulation, FIGS. 19A-19H display individual organ data for both sexes, enabling direct comparison and identification of any sex-dependent differences in biodistribution.

[0130] Mice were euthanized and muscle tissue (site of injection), liver, kidney, spleen, draining lymph nodes, lung, heart, and brain were collected and small chunks were weighed into previously weight microcentrifuge tubes so the weight of the tissue was known. 100 pL of IX Triton was added to each tissue (except lymph nodes where 50 pL was added) and the tissues were frozen at -80 °C overnight. In the morning, tissues were homogenized with plastic pestle which fits into 1.5 mL microcentrifuge tubes. Pestle was rinsed with water and dried between tissue samples. After homogenization, the samples were left at 4 °C for two hours before they were again frozen overnight at -80 °C to further break up the tissue. In the morning, samples were centrifuged for 10 minutes at 14,000 x G. 10 pL of supernatant was added to a new transparent microcentrifuge tube followed by 50 pL of ProMega luciferin assay reagent immediately prior to reading the luminescence on a ProMega GLOMAX 20 / 20 Luminometer. Luminescence was normalized to the mass of tissue.

[0131] In muscle expression, similar to the pattern observed with full-body luminescence, the shorter polymers appear to outperform the DP20 variants. In both females and males, the PMeOx-DPIO polymer showed significantly higher expression compared to all other polymers (FIG. 19A). In the liver (FIG. 19B), expression levels are relatively consistent across formulations, with no dramatic differences. However, in females, PEtOx-DP20 exhibits statistically higher liver expression than the other formulations. This trend is not observed in males, where only PMeOx-DPIO shows higher expression than PMeOx-DP20. Notably, liver expression values remain within the same order of magnitude across all formulations, whereas muscle expression spans multiple orders of magnitude. In the kidney (FIG. 19C), PMeOx-DPIO demonstrates higher expression in males compared to all other groups, while no significant differences are observed among females.

[0132] A key finding in this example is the differential expression observed in the spleen and lymph nodes (FIG. 19D and FIG. 19E). These organs serve as critical training grounds for the immune system, and enhancing expression in these areas is likely to improve vaccine efficacy and therapeutic outcomes. In females, the shorter DP 10 polymers again demonstrate superior transfection efficiency, with PEtOx-DPIO showing significantly higher expression than most other polymers. In the lymph nodes, PMeOx-DPIO outperforms some formulations, and although no statistically significant differences are observed in males, PMeOx-DPIO still exhibits the highest transfection efficiency.

[0133] Overall, LNPs with the shorter PMeOx and PEtOx DP 10 polymers demonstrate more effective tissue permeation compared to their longer-chain counterparts. This observation aligns with the full-body luminescent imaging results, which revealed broader tissue distribution for the shorter polymers. As a result, these formulations achieve higher expression levels in a wider range of tissues than both PEG-2000 LNPs and POx lipids with longer polymer chains.

[0134] EXAMPLE 7 - Freeze / Thaw Stability of Lipid Nanoparticles LNP formulations containing EtOx-20 LNPs were frozen with and without different cryoprotectants and in vitro transfection in HEK cells was compared to fresh (never frozen) formulations. The results are provided in FIG. 20A. MeOx-DPIO and DP20 formulations with glycerol (5%) and / or sucrose (9%) cryoprotection were compared to standard PEG formulations. In vitro transfection in HEK cells was compared to fresh (never frozen) formulations. The results are provided in FIG. 20B.

[0135] EXAMPLE 8 - Deprotection of MestOx Polymer

[0136] MestMe polymer of FIG. 21 was produced according to cationic ring opening polymerization as described herein. The polymer was refluxed in NaOH overnight to remove the methyl ester, thereby yielding carboxylate on the functional block. Peak position A in FIG. 21 disappears confirming production of the carboxylate.

[0137] EXAMPLE 9 - Amidation of MestOx Polymer

[0138] MestMe polymer was refluxed in ethanolamine overnight. Amidation was confirmed by disappearance of methyl ester peaks (A) and the appearance of flaking peaks (B) indicative of ethanolamine conjugation, as illustrated in FIG. 22.

[0139] EXAMPLE 10 - Amidation of MestOx Polymer

[0140] MestMe polymer was refluxed in mopholino propyl amine overnight. Amidation was confirmed by disappearance of methyl ester peaks (A) and the appearance of a peak associated with the morpholine propyl amine group, as illustrated in FIG. 23.

[0141] EXAMPLE 11 - Synthesis of Polymethyl Oxazine

[0142] Polymethyl oxazine was synthesized via cationic ring opening polymerization of 2- methyl-2-oxazine monomer with the use of sodium azide as the terminating agent. FIG. 24 confirms the synthesis of the azide terminated polymethyl oxazine.

[0143] EXAMPLE 12 - Conjugate Synthesis

[0144] Polymer from Examples 8, and 10-11 were coupled to PE-DBCO to provide conjugates described herein. The coupling reactions were monitored via the absorbance of free DBCO groups at 310 nm, wherein decrease in the DBCO absorbance evidences the coupling of lipid and polymer. FIGS. 25 illustrates UV-VIS spectra monitored at 310 nm confirming the coupling of the polymers with the DBCO-PE phopsholipids.

[0145] EXAMPLE 13 - Lipid Nanoparticle Synthesis

[0146] Conjugates synthesized according to Example 12 were formed into lipid nanoparticles according to the procedure of Example 3. Properties of the resultant lipid nanoparticles are provided in Table 4.

[0147] Table 4 - Lipid Nanoparticle Properties

[0148] EXAMPLE 14 - Jn Vitro Transfection

[0149] In vitro transfection of HEK cells was conducted with the lipid nanoparticles of Example 13 in accordance with the protocol of Example 4. FIG. 26 provides the results of the transfection study.

[0150] EXAMPLE 15 - In Vivo Administration and Ex-Vivo Analysis

[0151] Batten disease mice were administered either MeOx-DPIO or EtOx-DP20 LNPs by IV injection of 5 ug of firefly luciferase mRNA contained in the LNPs and compared to standard PEG formulation. After 24 hours, mice were euthanized, organs harvested, and luminescence analyzed and normalized to organ weight according to the protocol of Example 6. Results of the study are provided in FIG. 27. As illustrated in FIG. 27, the data indicated that oxazoline LNPs have less liver and kidney accumulation and improved accumulation in the lymph nodes compared to standard PEG formulations.

[0152] EXAMPLE 16 - Lipid Nanoparticles Incorporating Small Molecules with Lipophilic Tail

[0153] In this example, an LNP is formulated using a POx lipid and containing also containing the RC16 derivative in the LNP. In this example, SM-102 ionizable lipid, cholesterol, DSPC, and POx-ylated lipid are mixed in a 50:38.5:10: 1.5 molar ratio. RC16 is dissolved in the organic phase with the lipids at various amounts and formulation proceeds according to prior described procedure. RC16 could be incorporated at a mass ratio ranging from 0.01-1.0 mg RC16 per 0.96 mg of lipids described above.

[0154] The integrated adjuvant does not need to be C16 modified, but can be cholesterol modified, or any other hydrophobic modification described in prior patent. Nor does the adjuvant need to be TLR 7 / 8 agonist / resiquimod.

[0155] EXAMPLE 17 - Biological Activity of Resiquimod derivatives w ilh Lipophilic Tail

[0156] A series of new hydrophobically modified derivatives of resiquimod to anchor the drug in the lipid shell of lipid nanoparticles described herein are synthesized (FIG. 4). In the compounds, alkyl or cholesteryl groups are linked to resiquimods’ functionally important amino group via carbamate link that is cleavable by in cells. Lipid nanoparticles incorporating the resiquimod derivates can be prepared in view of Example 16. The biological activity of new prodrugs is confirmed by measuring the TNF-Alpha expression in RAW264.7 macrophages.

[0157] As will be understood by those familiar with the art, the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the disclosures and descriptions herein are intended to be illustrative, but not limiting, of the scope of the invention which is set forth in the following claims.

Claims

CLAIMS1. A conjugate comprising: a lipid coupled to a hompolymer via a linker, L, wherein the homopolymer is of formula(I):wherein R1is selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2, and n is 5 to 500.

2. The conjugate of claim 1, wherein R1is -C(O)OR2.

3. The conjugate of claim 2, wherein monomer of the homopolymer is selected from the group consisting of 2-methoxycarboxyethyl-2-oxazoline, 2-carboxyethyl-2-oxazoline, 2- methoxycarboxyethyl-2-oxazine, and 2-carboxyethyl-2-oxazine.

4. A conjugate comprising: a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II):wherein R1is selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10.

5. The conjugate of claim 4, wherein the copolymer is a random copolymer.

6. The conjugate of claim 4, wherein the monomer of formula (II) has a gradient distribution in the copolymer.

7. The conjugate of claim 6, wherein a majority of the monomer of formula (II) is located distal from the lipid.

8. The conjugate of claim 6, wherein a majority of the monomer of formula (II) is located proximate the lipid.

9. The conjugate of claim 1, wherein the hydrophilic component includes one or more blocks comprising the oxazoline monomer or oxazine monomer.

10. The conjugate of claim 9, wherein the functional component includes one or more blocks comprising the monomer of formula (II).

11. The conjugate of claim 10, wherein the functional component is located distal to the lipid.

12. The conjugate of claim 10, wherein the functional component is located proximate the lipid.

13. The conjugate of any of claims 4 to 12, wherein the copolymer has a number average degree of polymerization of 5 to 500.

14. The conjugate of claim 4, wherein the functional component comprises 1 to 15 monomers of formula (II).

15. The conjugate of any of claims 4 to 12, wherein the oxazoline monomer is selected from the group consisting of 2-methyl-2-oxazoline, 2-ethyl-2-oxazoline, 2-propyl-2-oxazoline, 2- isopropyl-2-oxazoline, 2-butyl-2-oxazoline, and combinations thereof.

16. The conjugate of any of claims 4 to 12, wherein the oxazine monomer 2-methyl-2- oxazine, 2-ethyl-2-oxazine, 2-propyl-2-oxazine, isopropyl-2-oxazine, 2-butyl-2-oxazine, and combinations thereof.

17. The conjugate of any of claims 4 to 12, wherein R1is -C(O)OR2.

18. The conjugate of claim 18, wherein the monomer of formula (II) is selected from the group consisting of 2-methoxycarboxyethyl-2-oxazoline, 2-carboxyethyl-2-oxazoline, 2- methoxycarboxyethyl-2-oxazine, 2-carboxyethyl-2-oxazine, and mixtures thereof.

19. The conjugate of any of claims 4 to 12, wherein the monomer of formula (II) accounts for 10 percent to 95 percent of the copolymer.

20. The conjugate of claim 4, wherein the lipid is selected from the group consisting of phospholipid, a cationic lipid, an anionic lipid, a ionizable lipid, a steroid compound such as cholesterol, or mixtures thereof.

21. The conjugate of claim 4, wherein the lipid comprises two hydrophobic tails.

22. The conjugate of claim 4, wherein the lipid is selected from the group consisting distearoylphosphoethanolamine (DPSE), diacylglyceral (DAG), dimyristoyl glycerol (DMG), cholestryl, and tocopheryl.

23. The conjugate of claim 4 further comprising a reactive moiety for attaching a targeting ligand at an end of the copolymer distal to the lipid.

24. The conjugate of claim 23, wherein the targeting ligand is selected from the group consisting of an aptamer, antibody, antibody fragment, peptide, and small molecule.

25. The conjugate of claim 1 or claim 4 further comprising a targeting ligand coupled to the monomer of formula (II).

26. The conjugate of claim 25, wherein the targeting ligand is selected from the group consisting of an aptamer, antibody, antibody fragment, peptide, and small molecule.

27. A lipid nanoparticle comprising: a conjugate including a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is of formula (I):wherein R1is selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2areoptionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2, and n is 5 to 500.

28. The lipid nanoparticle of claim 27, wherein R1is -C(O)OR2.

29. The lipid nanoparticle of claim 28, wherein monomer of the homopolymer is selected from the group consisting of 2-methoxycarboxyethyl-2-oxazoline, 2-carboxyethyl-2-oxazoline, 2-methoxycarboxyethyl-2-oxazine, and 2-carboxyethyl-2-oxazine.

30. The lipid nanoparticle of claim 27 further comprising at least one of ionizable lipids and helper lipids.

31. The lipid nanoparticle of claim 30, wherein the helper lipids comprise structural ligands, cholesterol, or mixtures thereof.

32. The lipid nanoparticle of claim 30, wherein the ionizable lipids comprises cationic lipids.

33. The lipid nanoparticle of claim 27 further comprising a biomolecular species and / or small molecule within the lipid nanoparticle interior.

34. The lipid nanoparticle of claim 33, wherein the biomolecular species is negatively charged.

35. The lipid nanoparticle of claim 34, wherein the biomolecular species comprises one or more nucleic acids, oligonucleotides, or mixtures thereof.

36. The lipid nanoparticle of claim 35, wherein the one or more nucleic acids are selected from the group consisting of DNA and RNA.

37. The lipid nanoparticle of claim 36, wherein the DNA comprises single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA), plasmid DNA (p-DNA), and mixtures thereof.

38. The lipid nanoparticle of claim 36, wherein the RNA comprises messenger RNA (mRNA), transfer RNA (RNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), circular RNA (circRNA), and small-activating RNA (sa-RNA).

39. The lipid nanoparticle of claim 35, wherein the one or more nucleic acids are naturally occurring.

40. The lipid nanoparticle of claim 35, wherein the one or more nucleic acids are synthetic.

41. A lipid nanoparticle comprising: a conjugate including a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II):wherein R1is selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10.

42. The lipid nanoparticle of claim 41 further comprising at least one of ionizable lipids and helper lipids.

43. The lipid nanoparticle of claim 42, wherein the helper lipids comprise structural ligands, cholesterol, or mixtures thereof.

44. The lipid nanoparticle of claim 42, wherein the ionizable lipids comprises cationic lipids.

45. The lipid nanoparticle of claim 41, wherein the conjugate is selected from any of claims 5 to 26.

46. The lipid nanoparticle of claim 41 further comprising a biomolecular species and / or small molecule within the lipid nanoparticle interior.

47. The lipid nanoparticle of claim 46, wherein the biomolecular species is negatively charged.

48. The lipid nanoparticle of claim 47, wherein the biomolecular species comprises one or more nucleic acids, oligonucleotides, or mixtures thereof.

49. The lipid nanoparticle of claim 48, wherein the one or more nucleic acids are selected from the group consisting of DNA and RNA.

50. The lipid nanoparticle of claim 49, wherein the DNA comprises single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA), plasmid DNA (p-DNA), and mixtures thereof.

51. The lipid nanoparticle of claim 49, wherein the RNA comprises messenger RNA (mRNA), transfer RNA (RNA), ribosomal RNA (rRNA), microRNA (miRNA), small interfering RNA (siRNA), circular RNA (circRNA), and small-activating RNA (sa-RNA).

52. The lipid nanoparticle of claim 48, wherein the one or more nucleic acids are naturally occurring.

53. The lipid nanoparticle of claim 48, wherein the one or more nucleic acids are synthetic.

54. A method of making a lipid nanoparticle comprising: providing a mixture including free lipids and a conjugate comprising a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is of formula (I):wherein R1is selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2, and n is 5 to 500; and forming the free lipids and the conjugate into a shell of the lipid nanoparticle, wherein the homopolymer resides on an exterior of the shell.

55. The method of claim 54, wherein the free lipids comprise at least one of ionizable lipids and helper lipids.

56. The method of claim 55, wherein the helper lipids comprise structural ligands, cholesterol, or mixtures thereof.

57. The method of claim 54, wherein the conjugate is selected from any of claims 1-3.

58. The method of claim 54, wherein the mixture further comprises one or more biomolecular species, and the biomolecular species is incorporated into an interior defined by the shell.

59. The method of claim 58, wherein the biomolecular species comprises one or more nucleic acids, oligonucleotides, or mixtures thereof.

60. The method of claim 59, wherein the one or more nucleic acids are selected from the group consisting of DNA and RNA.

61. The method of claim 60, wherein the DNA comprises single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA), plasmid DNA (p-DNA), and mixtures thereof.

62. The method of claim 60, wherein the RNA comprises messenger RNA (mRNA), transfer RNA (RNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), microRNA (miRNA) circular RNA (circRNA), and small-activating RNA (sa-RNA).

63. A method of making a lipid nanoparticle comprising: providing a mixture including free lipids and a conjugate comprising a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II):wherein R1is selected from the group consisting hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10; andforming the free lipids and the conjugate into a shell of the lipid nanoparticle, wherein the copolymer resides on an exterior of the shell.

64. The method of claim 63, wherein the free lipids comprise at least one of ionizable lipids and helper lipids.

65. The method of claim 64, wherein the helper lipids comprise structural ligands, cholesterol, or mixtures thereof.

66. The method of claim 63, wherein the conjugate is selected from any of claims 5 to 26.

67. The method of claim 63, wherein the mixture further comprises one or more biomolecular species, and the biomolecular species is incorporated into an interior defined by the shell.

68. The method of claim 67, wherein the biomolecular species comprises one or more nucleic acids, oligonucleotides, or mixtures thereof.

69. The method of claim 68, wherein the one or more nucleic acids are selected from the group consisting of DNA and RNA.

70. The method of claim 69, wherein the DNA comprises single-stranded DNA (ss-DNA), double-stranded DNA (ds-DNA), plasmid DNA (p-DNA), and mixtures thereof.

71. The method of claim 69, wherein the RNA comprises messenger RNA (mRNA), transfer RNA (RNA), ribosomal RNA (rRNA), small interfering RNA (siRNA), microRNA (miRNA) circular RNA (circRNA), and small-activating RNA (sa-RNA).

72. A lipid nanoparticle comprising: a conjugate including a lipid coupled to a homopolymer via a linker, L, wherein the homopolymer is of formula (I):wherein R1is selected from the group consisting of alkyl and cycloalkyl, the alkyl and cycloalkyl each substituted with at least one substituent selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl, and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2, and n is 5 to 500; and a small molecule encapsulated by the lipid nanoparticle, the small molecule comprising a lipid or lipophilic tail interacting with a lipid shell of the lipid nanoparticle.

73. The lipid nanoparticle of claim 72, wherein the small molecule is operable to interact with one or more immune receptors.

74. A lipid nanoparticle comprising: a conjugate including a lipid coupled to a copolymer via a linker, L, wherein the copolymer comprises a hydrophilic component comprising oxazoline monomer or oxazine monomer, and a functional component comprising monomer of formula (II):wherein R1is selected from the group consisting of hydrogen, hydroxyl, morpholine, nucleotide, nucleoside, -SH, -C(O)OR2, and C(O)NR3R4, wherein R2is selected from hydrogen and alkyl,and R3and R4are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl, wherein the alkyl, cycloalkyl, amine, aryl, heteroaryl, and heterocyclyl of R3and R4and the alkyl of R2are optionally substituted with one or more substituents selected from the group consisting of hydroxyl, halo, amine, and NO2, and wherein m is 1 or 2 and p ranges from 0 to 10; and and a small molecule encapsulated by the lipid nanoparticle, the small molecule comprising a lipid or lipophilic tail interacting with a lipid shell of the lipid nanoparticle.

75. The lipid nanoparticle of claim 74, wherein the small molecule is operable to interact with one or more immune receptors.

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