Poly(oxazoline) conjugates with pendant-type cationic groups, and lipid nanoparticles and polyplexes containing them.

Abstract

Poly(oxazoline) conjugates (cationic POZs) with pendant-type cationic groups used to facilitate the delivery of encapsulated payloads, and lipid nanoparticles (LNPs) containing cationic POZs are disclosed. LNPs and polyplexes containing cationic POZs and nucleic acid payloads, for example, mRNA or modified mRNA, are disclosed. Such LNPs are immunogenic or have reduced immunogenicity compared to corresponding LNPs containing ionizable lipids.

Description

[Technical Field]

[0001] This disclosure relates to polyoxazoline (POZ) conjugates with pendent-type terminal cationic groups (cationic POZs) and methods for their synthesis. Furthermore, this disclosure relates to lipid nanoparticles (LNPs) and polyplexes containing cationic POZs, and pharmaceutical compositions containing such LNPs. LNPs and polyplexes incorporating oligonucleotides such as mRNA, DNA, saRNA, and siRNA for delivery to living cells are also envisioned. [Background technology]

[0002] Nucleic acid (especially mRNA)-based vaccines offer advantages over other vaccine technologies. Such vaccines can be produced with reduced development time and cost by utilizing commonly used manufacturing platforms based on lipid nanoparticles (LNPs). Indeed, the usefulness of LNPs for delivering mRNA to cells was demonstrated in the COVID-19 (or SARS-CoV-2) vaccine that addressed the global pandemic that resulted in over 7 million deaths worldwide. Given the enormous burden on healthcare systems caused by this viral disease, and the widespread utility of mRNA cell delivery for a variety of diseases ranging from cancer to influenza, there has been extensive research into understanding and improving RNA LNP delivery.

[0003] As shown in Figure 1, the basic structure of LNPs is realized by four components: (1) sterol lipids (which provide flexibility, e.g., cholesterol), (2) helper lipids (which provide structural integrity, e.g., distearoylphosphatidylcholine (DSPC)), (3) polymer lipids (which stabilize lipid nanoparticles and prevent fusion with other nanoparticles, e.g., PEG-lipids or POZ-lipids), and (4) ionizable lipids (which complex with oligonucleotides). Most commonly, these components are prepared in two separate phases: 1) an aqueous phase (containing RNA or other oligonucleotides) in a buffer at pH approximately 4-5, and 2) an organic phase containing cholesterol / DSPC / polymer-lipids / ionizable lipid components in ethanol. By utilizing microfluidic techniques in which the flow rates of these two phases are precisely controlled, the delivery of nucleic acid payloads can be facilitated to prepare LNPs of the desired size that produce a therapeutic response.

[0004] In this embodiment, ionizable lipids have been shown to be crucial components of LNPs. However, preclinical studies have linked ionizable lipids to a number of undesirable side effects in patients, ranging from enhanced reactivity (adverse events) to blood coagulation abnormalities, suggesting that these side effects include binding of PEG-lipid nanoparticles following immunization with approved mRNA vaccines, activation of complement activation pathways, introduction of pro-inflammatory pathways, and accelerated LNP clearance. Therefore, the properties of ionizable lipids can have a significant impact on payload delivery and expression (for example, MC3 works exceptionally well for siRNA delivery but generally poorly for mRNA delivery), and the art requires the development of technologies to reduce or eliminate undesirable side effects to repeated dose administration that may be associated with ionizable lipids. More specifically, the art requires non-immunogenic (or at least significantly reduced immunogenic) alternatives to ionizable lipids that do not impair the efficacy of LNPs.

[0005] Synthetic cationic polymers are used as non-viral vectors in gene therapy. In this embodiment, long DNA strands are broken down into nanoparticles of 50-300 nm in size by condensation with polyamines such as poly(L-lysine), spermidine, and spermine (Thomas, TJ, Heidar-Ali Tajmir-Riahi, and CKS Pillai (2019), "Biodegradable Polymers for Gene Delivery," Molecules, vol. 24, no. 20, p. 3744. https: / / doi.org / 10.3390 / molecules24203744). However, highly positively charged polymers containing primary amines have high zeta potentials and can cause cytotoxicity.

[0006] Polymer chains containing secondary amines have also been used, including linear and branched polyethyleneimine (PEI) polymers. These polymers have been shown to have high transfection efficiency. For example, U.S. Patent No. 11,318,195 describes polyplexes with an N / P ratio of 2-15, made from PEI with a molecular weight of 5,000-25,000 Da and single-stranded RNA. The toxicity of free, pure PEI to HEK-293 cells was observed at 77 μM IC50. 50 To reduce these toxic effects, these polymers were copolymerized with inert polymers such as polyethylene glycol (PEG), polysaccharides, dextran, polycaprolactone, and polyglutamic acid.

[0007] Copolymers of poly(methyloxazoline) and poly(decenyloxazoline) or poly(butenyloxazoline) have been discussed. See, for example, Rinkenauer AC et al. (March 2015): A Cationic Poly(2-oxazoline) with High In Vitro Transfection Efficiency Identified by a Library Approach; Macromolecular Bioscience, vol. 15, no. 3, pp. 414-425 (https: / / doi.org / 10.1002 / mabi.201400334). In this embodiment, a "longer" decenyl or "shorter" butenyl side chain is attached to the thiolamine by photoaddition to create a charged oxazoline polymer having a primary or tertiary thioether amine. The polymer size is approximately 20,000 Da to 30,000 Da, and the polydispersity index (PDI) is approximately 1.4. These polymers were condensed with the plasmid pEGFP-N1 (4.7kb) to produce polyplexes with sizes of 90–200 nm and zeta potentials of -4–+40 mV. However, the polyplexes exhibited very low transfection properties (less than 25%). Furthermore, these polymers were found to be unsafe and to cause hemolysis of red blood cells.

[0008] In addition to the safety concerns of cationic polymers and polyplexes described above, there have been no attempts to prepare LNPs from these polymers or polyplexes. This disclosure provides solutions to the shortcomings of the prior art. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] U.S. Patent No. 11,318,195 [Non-patent literature]

[0010] [Non-Patent Document 1] Thomas, TJ, Heidar-Ali Tajmir-Riahi, and CKS Pillai (2019), "Biodegradable Polymers for Gene Delivery", Molecules, Vol. 24, No. 20, p. 3744. https: / / doi.org / 10.3390 / molecules24203744 [Non-Patent Document 2] Rinkenauer AC et al. (March 2015): A Cationic Poly(2-oxazoline) with High In Vitro Transfection Efficiency Identified by a Library Approach; Macromolecular Bioscience, Vol. 15, No. 3, pp. 414-425 (https: / / doi.org / 10.1002 / mabi.201400334) [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] (Summary of the invention) This disclosure relates to polyoxazoline (POZ) conjugates with pendent-terminal cationic groups (cationic POZs) that are suitable as alternatives to ionizable lipids used in commercially available mRNA vaccines. In particular, the inventors have made the surprising observation that LNPs and / or polyplexes prepared with cationic POZs result in efficient and therapeutic delivery of RNA and DNA payloads. [Means for solving the problem]

[0012] In some embodiments, the cationic POZ is cationic poly(ethyl oxazoline) (cationic PEOZ). In this embodiment, LNPs comprising sterol lipids, helper lipids, polymer lipids, and cationic PEOZ can be prepared according to the disclosure (cationic PEOZ LNPs). In some embodiments, the polymer lipids can be POZ-lipids. For example, cationic PEOZ LNPs can be prepared from cholesterol, DSPC, POZ-lipids, and cationic PEOZ. Similarly, polyplexes can be prepared from cationic POZ and payloads.

[0013] In this invention, poly(ethyl oxazoline) copolymers with smaller molecular weights of 1,000 to 3,000 Da and PDI of less than 1.2 were prepared using pentinyl oxazoline (PtynOZ) and methyl oxazolinyl propionate (MeEstOZ) monomers. Cationic POZ polymers having spermidine, spermine, choline, and N-methylpiperidine groups were prepared using these smaller polymers, and LNPs for delivering RNA and DNA payloads were prepared using these polymers.

[0014] This disclosure is, Cationic POZ, Helper lipids, Polymer lipids, and Sterol lipids Lipid nanoparticles containing a cationic POZ of formulas I-IV: R-POZ1-cation (I) [In the formula, R comprises an initiator, and POZ1 comprises a polyoxazoline polymer.]

[0015] [ka] [In the formula, R includes an initiator group, n ranges from 1 to 10, R2 is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups for each repeating unit, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.]

[0016] [ka] [In the formula, R includes an initiator group, n ranges from 1 to 10, R2 is independently selected for each repeating unit from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.], and

[0017] [ka] [In the formula, R includes an initiator, n ranges from 1 to 10, R2 is independently selected for each repeating unit from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, m ranges from 1 to 100, a ranges from 3 to 20, and T includes a terminal group.] This also relates to lipid nanoparticles selected from the group consisting of one of the following.

[0018] In some embodiments, the lipid nanoparticles contain about 0.1% to about 10% cationic POZ, about 30% to about 80% sterol lipids, about 0.5% to about 20% polymer lipids, and about 15% to about 65% helper lipids. In other embodiments, the lipid nanoparticles contain about 30% to about 70% cationic POZ, about 30% to about 50% sterol lipids, about 0.5% to about 20% polymer lipids, and about 5% to about 15% helper lipids.

[0019] In yet another embodiment, the polymer lipid is a POZ-lipid of formula V: R-POZ2-L-lipid (V) [In the formula, R includes an initiator, POZ2 contains poly(oxazoline), L contains a linking group, The lipids include uncharged lipids containing at least one hydrophobic moiety.

[0020] In some embodiments, the linking group is physiologically degradable. In other embodiments, the linking group is stable.

[0021] This disclosure is, Cationic POZ of formula I R-POZ1-cation (I) [In the formula, R comprises an initiator, and POZ1 comprises a polyoxazoline polymer.] Helper lipids, Polymer lipids, and Sterol lipids This also relates to lipid nanoparticles containing these nanoparticles.

[0022] In some embodiments, the polymer lipid is a POZ-lipid of formula V: R-POZ2-L-lipid (V) [In the formula, R includes an initiator, POZ2 contains poly(oxazoline), L contains a linking group, The lipids include uncharged lipids containing at least one hydrophobic moiety.

[0023] In some embodiments, the linking group is physiologically degradable. In other embodiments, the linking group is stable.

[0024] In other embodiments, the lipid nanoparticles contain about 0.1% to about 10% cationic POZ, about 30% to about 80% sterol lipids, about 0.5% to about 20% polymer lipids, and about 15% to about 65% helper lipids. In yet another embodiment, the lipid nanoparticles contain about 30% to about 70% cationic POZ, about 30% to about 50% sterol lipids, about 0.5% to about 20% polymer lipids, and about 5% to about 15% helper lipids.

[0025] This disclosure is, Cationic POZ of formula II:

[0026] [ka] [In the formula, R includes an initiator group, n ranges from 1 to 10, R2 is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups for each repeating unit, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.] Helper lipids, Polymer lipids, and Sterol lipids This also relates to lipid nanoparticles containing these nanoparticles.

[0027] In some embodiments, the polymer lipid is a POZ-lipid of formula V: R-POZ2-L-lipid (V) [In the formula, R includes an initiator, POZ2 contains poly(oxazoline), L contains a linking group, The lipids include uncharged lipids containing at least one hydrophobic moiety.

[0028] In some embodiments, the linking group is physiologically degradable. In other embodiments, the linking group is stable.

[0029] In other embodiments, the lipid nanoparticles contain about 0.1% to about 10% cationic POZ, about 30% to about 80% sterol lipids, about 0.5% to about 20% polymer lipids, and about 15% to about 65% helper lipids. In yet another embodiment, the lipid nanoparticles contain about 30% to about 70% cationic POZ, about 30% to about 50% sterol lipids, about 0.5% to about 20% polymer lipids, and about 5% to about 15% helper lipids.

[0030] This disclosure is, Cationic POZ of formula III:

[0031] [ka] [In the formula, R includes an initiator group, n ranges from 1 to 10, R2 is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups for each repeating unit, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.] Helper lipids, Polymer lipids, and Sterol lipids This also relates to lipid nanoparticles containing these nanoparticles.

[0032] In some embodiments, the polymer lipid is a POZ-lipid of formula V: R-POZ2-L-lipid (V) [In the formula, R includes an initiator, POZ2 contains poly(oxazoline), L contains a linking group, The lipids include uncharged lipids containing at least one hydrophobic moiety.

[0033] In some embodiments, the linking group is physiologically degradable. In other embodiments, the linking group is stable.

[0034] In other embodiments, the lipid nanoparticles contain about 0.1% to about 10% cationic POZ, about 30% to about 80% sterol lipids, about 0.5% to about 20% polymer lipids, and about 15% to about 65% helper lipids. In yet another embodiment, the lipid nanoparticles contain about 30% to about 70% cationic POZ, about 30% to about 50% sterol lipids, about 0.5% to about 20% polymer lipids, and about 5% to about 15% helper lipids.

[0035] This disclosure is, Cationic POZ of formula IV:

[0036] [ka] [In the formula, R includes an initiator, n ranges from 1 to 10, R2 is independently selected for each repeating unit from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, m ranges from 1 to 100, a ranges from 3 to 20, and T includes a terminal group.] Helper lipids, Polymer lipids, and Sterol lipids This also relates to lipid nanoparticles containing these nanoparticles.

[0037] In some embodiments, the polymer lipid is a POZ-lipid of formula IV: R-POZ2-L-lipid (IV) [In the formula, R includes an initiator, POZ2 contains poly(oxazoline), L contains a linking group, The lipids include uncharged lipids containing at least one hydrophobic moiety.

[0038] In some embodiments, the linking group is physiologically degradable. In other embodiments, the linking group is stable.

[0039] In other embodiments, R includes glycerol, pentaerythritol, polyglycerol, or a combination thereof. In yet another embodiment, the lipid nanoparticles contain about 0.1% to about 10% cationic POZ, about 30% to about 80% sterol lipids, about 0.5% to about 20% polymer lipids, and about 15% to about 65% helper lipids. In yet another embodiment, the lipid nanoparticles contain about 30% to about 70% cationic POZ, about 30% to about 50% sterol lipids, about 0.5% to about 20% polymer lipids, and about 5% to about 15% helper lipids.

[0040] This disclosure also relates to compounds of formula I: R-POZ1-cation (I) [In the formula, R comprises an initiator, and POZ1 comprises a polyoxazoline polymer.] In some embodiments, R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, or a substituted or unsubstituted aralkyl group.

[0041] This disclosure also relates to compounds of formula II:

[0042] [ka] [In the formula, R includes an initiator group, n ranges from 1 to 10, R2 is independently selected for each repeating unit from an unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional group, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.] In some embodiments, R includes hydrogen, a substituted or unsubstituted alkyl, an alkyne-substituted alkyl, a triazole having a bonded carboxylic acid, or a substituted or unsubstituted aralkyl group.

[0043] This disclosure also relates to compounds of formula III:

[0044] [ka] [In the formula, R includes an initiator group, n ranges from 1 to 10, R2 is independently selected for each repeating unit from an unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional group, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.] In some embodiments, R includes hydrogen, a substituted or unsubstituted alkyl, an alkyne-substituted alkyl, a triazole having a bonded carboxylic acid, or a substituted or unsubstituted aralkyl group.

[0045] This disclosure also relates to compounds of formula IV:

[0046] [ka] [In the formula, R includes an initiator, n ranges from 1 to 10, R2 is independently selected for each repeating unit from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, m ranges from 1 to 100, a ranges from 3 to 20, and T includes a terminal group.] In some embodiments, R includes glycerol, pentaerythritol, polyglycerol, or a combination thereof.

[0047] Further features and effects of the present invention can be confirmed from the following detailed description, presented in conjunction with the drawings described below. [Brief explanation of the drawing]

[0048] [Figure 1] This is a diagram showing typical lipid nanoparticles. [Figure 2] This figure shows lipid nanoparticles according to the disclosed embodiment. [Figure 3A] This figure shows the payload bound to the cationic POZ at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 3B] This figure shows the payload bound to the cationic POZ at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 4A] This figure shows a payload bonded to a cationic POZ with different linkers at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 4B] This figure shows a payload bonded to a cationic POZ with different linkers at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 5A] This figure shows the payload bound to the cationic POZ at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 5B] This figure shows the payload bound to the cationic POZ at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 6A] This figure shows a payload bonded to a cationic POZ with different linkers at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 6B] This figure shows a payload bonded to a cationic POZ with different linkers at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 7] This figure shows the transfection of HEK293 cells with LNPs in various ratios, doses, and buffers prepared in accordance with this disclosure. [Figure 8] This figure shows the transfection of HEK293 cells with LNPs in various ratios, doses, and buffers prepared in accordance with this disclosure. [Figure 9A] This figure shows the payload bound to the cationic POZ at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 9B] This figure shows the payload bound to the cationic POZ at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 9C]This figure shows the payload bound to the cationic POZ at increasing polymer concentrations, according to embodiments of the present disclosure. [Figure 10] This figure shows a microfluidic mixing system suitable for mixing a payload, lipids, and cationic POZ according to embodiments of the present disclosure. [Figure 11A] This figure shows the particle size of LNPs formed according to the embodiments of this disclosure. [Figure 11B] This figure shows the particle size of LNPs formed according to the embodiments of this disclosure. [Figure 12A] This figure shows the zeta potential of an LNP formed according to the embodiments of this disclosure. [Figure 12B] This figure shows the zeta potential of an LNP formed according to the embodiments of this disclosure. [Figure 13] This figure shows the particle size of the polyplex formed according to the embodiments of this disclosure. [Modes for carrying out the invention]

[0049] This disclosure provides cationic poly(oxazoline) (cationic POZ) suitable for replacing ionizable lipids in LNPs. In particular, cationic POZ LNPs can be prepared as shown in Figure 2 and may contain cationic POZ, sterol lipids, helper lipids, and polymer lipids. Cationic POZ LNPs prepared according to this disclosure can be used to preferentially deliver encapsulated payloads, such as nucleic acid payloads including, but not limited to, mRNA or modified mRNA.

[0050] The cationic POZ and other components of the cationic POZ LNP are described in more detail below.

[0051] definition All patent applications, patents, and printed publications cited herein are incorporated herein in their entirety by reference, except to the extent that any definition, waiver or denial of subject matter, and incorporated material does not correspond to the express disclosure herein, in which case the language in this disclosure shall prevail.

[0052] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, “an element” means one or more elements.

[0053] The terms "about" and "approximately" generally mean the acceptable range of error or variation in a measured quantity, taking into account the nature or precision of the measurement. Quantities described herein are approximations unless otherwise stated, and the terms "about" or "approximately" mean that they can be inferred unless otherwise specified.

[0054] In this specification, the terms “active” or “activated” refer to a functional group that readily reacts with an electrophile or nucleophile of another molecule, when used in relation to a particular functional group. This is in contrast to groups that require a catalyst or unrealistic reaction conditions to react (i.e., “unreactive” or “inactive” groups).

[0055] In this specification, the terms “physiologically degradable” or “physiologically releaseable” refer to a linkage containing a cleavable portion. The terms “degradable” and “releaseable” do not imply any specific mechanism by which the linker is cleaved.

[0056] In this specification, the terms “linking,” “linked,” “linkage,” or “linker” refer to a bond that is typically formed as a result of a chemical reaction when used with POZ polymers, POZ conjugates, activators, or compounds described herein, or components thereof, and are typically covalent bonds.

[0057] In this specification, the term “lipid nanoparticle” or “LNP” is used to encompass any of many types of nanoparticles, including liposomes, which are formed by a lipid layer surrounding a core containing molecules to be released into the body. Liposomes generally have one or more adjacent lipid bilayers encapsulating an aqueous core. Other forms of liposome-like nanocarriers may have a lipid monolayer or non-adjacent bilayers and may or may not have an aqueous core.

[0058] In this specification, the term "hydrophilic" refers to a compound or molecule, or a part thereof, in which the interaction with water is thermodynamically more favorable than the interaction with oil or other hydrophobic solvents, for example, with respect to a hydrophilic group. Hydrophilic compounds can be dissolved in or dispersed in water.

[0059] In this specification, the term "hydrophobic" refers to a compound or molecule, or a part thereof, in which, for example with respect to the hydrophobic portion, the interaction with water is thermodynamically less favorable than the interaction with oil or other hydrophobic solvents. Hydrophobic compounds can be dissolved or dispersed in oil or other hydrophobic solvents.

[0060] In this specification, the terms “inert” or “non-reactive,” when used in relation to a particular functional group, refer to a functional group that does not readily react with an electrophile or nucleophile of another molecule and requires a catalyst or unrealistic reaction conditions to react.

[0061] In this specification, the term "pendent group" refers to the portion of the POZ polymer that is bonded to the POZ polymer.

[0062] In this specification, the term “pendent moiety” refers to a substituent linked to the POZ polymer moiety via a linking group. The pendent moiety is exemplified by R2 in Formula IV as described herein.

[0063] In this specification, the term "pharmaceutically acceptable" means a compound that is compatible with other components of a composition and is not harmful to the compound or to the subject receiving the composition. In some embodiments, the term "pharmaceutically acceptable" means that it is approved by a U.S. federal or state regulatory authority or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias for use in animals, and more particularly in humans.

[0064] In this specification, the term “pharmaceutically acceptable form” includes, but is not limited to, known forms of a compound or POZ conjugate that can be administered to a subject, including solvates, hydrates, prodrugs, isomorphs, polymorphs, pseudomorphs, neutral forms, and salt forms of the compound. In some embodiments, the pharmaceutically acceptable form excludes prodrugs, isomorphs, and / or pseudomorphs. In some embodiments, the pharmaceutically acceptable form is limited to pharmaceutically acceptable salts, neutral forms, solvates, and hydrates. In some embodiments, the pharmaceutically acceptable form is limited to pharmaceutically acceptable salts and neutral forms. In some embodiments, the pharmaceutically acceptable form is limited to pharmaceutically acceptable salts.

[0065] In this specification, the term "alkyl," whether used alone or as part of a substituent, is a technical term and refers to a saturated aliphatic group that optionally contains one or more heteroatoms (such as O, S, or N) which may optionally be substituted, including linear alkyl groups, branched alkyl groups, cycloalkyl groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, a linear or branched alkyl group has about 30 or fewer carbon atoms in its main chain (for example, C1-C1 in a linear group). 30 In branched chains, C3~C 30 ), or having about 20 or fewer carbon atoms or 10 or fewer. In some embodiments, the term "alkyl" means C1-C 10 This refers to a linear alkyl group or a C1-C3 linear alkyl group. In some embodiments, the term "alkyl" means C3-C 12This refers to branched alkyl groups. In some embodiments, the term "alkyl" refers to C3-C8 branched alkyl groups. Representative examples of alkyl groups 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 C1-C8 branched alkyl groups containing one or more heteroatoms (such as O, S, or N) which may be optionally substituted in place of carbon atoms. 10 This refers to a linear alkyl group. In some embodiments, the term "alkyl" refers to a C1-C alkyl group that is substituted with up to five groups selected from the group consisting of OH, NH2, and =O. 10 This refers to a linear alkyl group.

[0066] In this specification, the term “alkenyl,” whether used alone or as part of a substituent, is a technical term referring to an unsaturated aliphatic group that optionally contains one or more heteroatoms (such as O, S, or N) which may optionally be substituted, including linear or branched hydrocarbon groups containing 2 to 30 carbon atoms and at least one carbon-carbon double bond formed by removing two hydrogen atoms. Representative examples of alkenyls 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 part and may have a (Z) or (E) stereoconfiguration with respect to the double bond.

[0067] In this specification, the term "alkynyl," whether used alone or as part of a substituent, is a technical term referring to an unsaturated aliphatic group containing 2 to 30 carbon atoms, including a linear or branched hydrocarbon group containing at least one carbon-carbon triple bond, and optionally containing one or more heteroatoms (such as O, S, or N) which may be optionally substituted. Representative examples of alkynyls include, but are not limited to, acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 4-pentynyl, and 1-butynyl.

[0068] In this specification, the terms “substituted alkyl,” “substituted alkenyl,” and “substituted alkynyl” refer to a group in which one or more bonds to carbon or hydrogen are replaced by bonds to non-hydrogen or non-carbon atoms, such as halogen atoms in halogens such as F, Cl, Br, I, etc. (but not limited to these); oxygen atoms in groups such as carbonyl, carboxyl, hydroxyl, alkoxy, aryloxy, heterocyclyloxy, and ester groups; sulfur atoms in groups such as thiol, alkyl and aryl sulfide groups, sulfone, sulfonyl, and sulfoxide groups; amines, amides, alkylamines, dialkylamines, arylamines, and arylamines. This refers to alkyl, alkenyl, and alkynyl groups as defined above, which are replaced by nitrogen atoms in groups such as alkylarylamines, diarylamines, N-oxides, imides, enamines, imines, oximes, hydrazones, heterocyclylamines, (alkyl)(heterocyclyl)amines, (aryl)(heterocyclyl)amines, diheterocyclylamines, triazoles, and nitriles; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and by bonding to 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 substituted with -OH groups and / or -C(O)-OH groups, such as C1-C5 alkyl, alkenyl, and alkynyl groups.

[0069] In this specification, the term "halo" or "halogen," whether used alone or as part of a substituent, is a technical term and refers to -F, -Cl, -Br, or -I.

[0070] In this specification, the term "alkoxy," whether used alone or as part of a substituent, is a technical term referring to an alkyl group as defined herein, which is attached to the parent molecule via an oxygen atom. Representative examples of alkoxys include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0071] In this specification, the terms "aralkyl" or "arylalkyl," whether used alone or as part of a substituent, are technical terms referring 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. "Substitutable aralkyl" has the same meaning as a substituted aryl group has with respect to an unsubstituted aryl group. However, a substituted aralkyl group also includes groups in which the carbon or hydrogen bond of the alkyl moiety is replaced by a bond to a non-carbon or non-hydrogen atom.

[0072] In this specification, the terms "heteroaralkyl" or "heteroarylalkyl," whether used alone or as part of a substituent, are technical terms referring to an alkyl group substituted with a heteroaryl group, which is attached to the parent molecule through the alkyl group. Heteroarylalkyls may be optionally substituted. The term "substituted heteroarylalkyl" has the same meaning as a substituted aryl group has with respect to an unsubstituted aryl group, in relation to an unsubstituted heteroarylalkyl group.

[0073] In this specification, the term “heterocyclylalkyl,” whether used alone or as part of a substituent, is a technical term referring 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 by a bond to a heterocyclyl group. Heterocyclylalkyls may be optionally substituted. The term “substituted heterocyclylalkyl” has the same meaning for an unsubstituted heterocyclylalkyl group as it does for a substituted aryl group. However, a substituted heterocyclylalkyl group also includes a group in which a non-hydrogen atom is bonded to a heteroatom in the heterocyclyl group of the heterocyclylalkyl group, for example, a nitrogen atom in the piperidine ring of a piperidinyl alkyl group, but is not limited to this.

[0074] In this specification, the term "aryl," whether used alone or as part of a substituent, is a technical term referring 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 halogens, azides, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, alkoxyls, aminos, nitros, sulfhydryls, iminos, amides, phosphonates, phosphinates, carbonyls, carboxyls, silyls, ethers, alkylthios, sulfonyls, sulfonamides, ketones, aldehydes, esters, heterocyclyls, aromatic or heteroaromatic moieties, fluoroalkyls (such as trifluoromethyl), and cyanos. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are shared between two adjacent rings (the rings are "fused rings"), where at least one of the rings is an aromatic hydrocarbon, and the other cyclic rings may be, for example, 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.

[0075] In this specification, the term "cycloalkyl," whether used alone or as part of a substituent, is a technical term referring to a saturated carbocyclic group containing 3 to 6 ring carbon atoms, where such rings may be optionally substituted with substituted or unsubstituted alkyl groups or substituents described for substituted alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-methylcyclobutyl, and 4-ethylcyclohexyl.

[0076] In this specification, the term "heteroaryl," whether used alone or as part of a substituent, is a technical term referring to monocyclic, bicyclic, and polycyclic aromatic groups having a total of 3 to 30 atoms, including one or more heteroatoms, such as nitrogen, oxygen, or sulfur, in their ring structure. 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 tropanil. A "heteroaryl" may be substituted with one or more substituents at one or more positions on the ring, such as halogens, azides, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, alkoxyls, aminos, nitros, sulfhydryls, iminos, amides, phosphonates, phosphinates, carbonyls, carboxyls, silyls, ethers, alkylthios, sulfonyls, sulfonamides, ketones, aldehydes, esters, heterocyclyls, aromatic or heteroaromatic moieties, fluoroalkyls (such as trifluoromethyl), cyanos, 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, for example, the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and / or heterocyclyls.

[0077] In this specification, the term "heterocyclyl," whether used alone or as part of a substituent, is a technical term and may be fully saturated or contain one or more unsaturated units. To avoid misunderstanding, the degree of unsaturation does not result in an aromatic ring system and refers to a non-aromatic ring system group having 3 to 15 atoms, including at least one heteroatom, e.g., nitrogen, oxygen, or sulfur, and including, but not limited to, monocyclic, bicyclic, and tricyclic rings. This should not be interpreted as limiting the scope of the present invention, but rather as illustrative examples, including the following: azilidinyl, azilinyl, oxylanil, thyranil, thyrenyl, dioxylanil, diazilinyl, diazepanil, 1,3-dioxanil, 1,3-dioxolanil, 1,3-dithiolanil, 1,3-dithianil, imidazolidinyl, isothiazolinil, isothiazolinil, isoxazolinil, isoxazolinil, azetyl, oxetanil, oxetyl, thietanil, thiethyl, diazetidinyl, dioxetanil, dioxetenyl, dithietanil, dithiethyl, dioxalanil, oxazolyl, thiazolyl, triazinyl, isothiazolyl, Isoxazolyl, azepine, azetidinil, morpholinil, oxadiazolinil, oxadiazolidinil, oxazolinil, oxozolidinil, oxopiperidinil, oxopyrrolidinil, piperazinil, piperidinil, pyranil, pyrazolinil, pyrazolidinil, pyrrolinil, pyrrolidinil, quinuclidinil, thiomorpholinil, tetrahydropyranil, tetrahydrofuranil, tetrahydrothienyl, thiadiazolinil, thiadiazolidinil, thiazolinil, thiazolidinil, thiomorpholinil, 1,1-dioxidethiomorpholinil (thiomorpholine sulfone), thiopyranil, and trithianil are examples of heterocyclic rings.The heterocyclyl group may be substituted at one or more positions on the ring with one or more substituents, such as halogens, azides, alkyls, aralkyls, alkenyls, alkynyls, cycloalkyls, hydroxyls, alkoxyls, aminos, nitros, sulfhydryls, iminos, amides, phosphonates, phosphinates, carbonyls, carboxyls, silyls, ethers, alkylthios, sulfonyls, sulfonamides, ketones, aldehydes, esters, heterocyclyls, aromatics or heteroaromatic moieties, fluoroalkyls (such as trifluoromethyl), cyanos, etc.

[0078] In this specification, the terms “treatment,” “to treat,” and “to treat” refer to a series of actions (such as administering a conjugate described herein or a pharmaceutical composition containing a conjugate described herein) to prevent, eliminate, or reduce the symptoms, aspects, or characteristics of a disease or condition. Such treatment does not need to be absolute in order to be useful. In one embodiment, treatment includes a series of actions initiated simultaneously with or after the onset of the symptoms, aspects, or characteristics of a disease or condition. In one embodiment, treatment includes a series of actions initiated before the onset of the symptoms, aspects, or characteristics of a disease or condition.

[0079] In this specification, the term “requiring treatment” means a judgment made by a caregiver that a patient requires or would benefit from treatment. This judgment is based on a variety of factors that are within the realm of 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 this disclosure.

[0080] In this specification, the terms “individual,” “subject,” or “patient” refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, and humans. Terms may specify male, female, or both, or may exclude male or female. In a preferred embodiment, the terms “individual,” “subject,” or “patient” refer to humans.

[0081] In this specification, the term “therapeutic dose” refers to the amount of conjugate alone or as part of a pharmaceutical composition that can have any detectable positive effect on any symptom, aspect, or characteristic of any symptom, aspect, or feature of any disease or condition. Such an effect does not need to be absolute to be beneficial.

[0082] It should be understood that the terms "substituted" or "substituted with" implicitly include the condition that such substitution results in a stable compound, subject to the allowable valencies of the substituted atom and substituent, and that the substitution does not spontaneously undergo transformation by rearrangement, fragmentation, decomposition, cyclization, elimination, or other reactions.

[0083] When a group is designated as a part of a compound, it will be understood that substitution of the group can be tailored to accommodate a particular bond. For example, when an alkyl group is linked to two other groups, the alkyl group can be considered an alkylene group.

[0084] The term “substituted” is also intended to encompass all permissible substituents of an organic compound. In a broad embodiment, permissible substituents include acyclic and cyclic, branched and unbranched substituents, carbocyclic and heterocyclyl substituents, aromatic and non-aromatic substituents of an organic compound. Exemplary substituents include, for example, those described above herein. In this disclosure, heteroatoms such as oxygen or nitrogen may have hydrogen substituents and / or any permissible substituents of the organic compound described herein that satisfy the valence of the heteroatom. Exemplary substitutions include, but are not limited to, hydroxy, 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 trifluoromethyl), cyano, and the like. This invention is not intended to be limited in any way by any permissible substituents on an organic compound.

[0085] Other chemical terms used herein are used in accordance with the common 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 scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0086] In this specification, 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. A pharmaceutically acceptable salt may include forms in which the ratio of molecules containing the salt is not 1:1. For example, a salt may contain more than one molecule of inorganic or organic acid per molecule of base, for example, two molecules of hydrochloric acid per molecule of conjugate. Alternatively, a salt may contain less than one molecule of inorganic or organic acid per molecule of base, for example, two molecules of conjugate per molecule of inorganic or organic acid.

[0087] In this specification, the terms “carrier” and “pharmaceutically acceptable carrier” refer to diluents, adjuvants, excipients, or vehicles to which a compound is administered or formulated together for administration. Non-limiting examples of such pharmaceutically 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. Furthermore, auxiliaries, stabilizers, viscosity modifiers, equipermants, cryopreservatives, lubricants, flavoring agents, and colorants 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), which are incorporated herein by reference in their entirety.

[0088] In this specification, the term “target molecule” means any molecule or compound having therapeutic or diagnostic application or targeting function, and refers to a vehicle to which it is administered or formulated for administration, and includes, but is not limited to, therapeutic agents (e.g., 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. The target molecule may form linkages with the active functional groups of the POZ polymers or POZ derivatives of this disclosure.

[0089] In this specification, “lipid” or “lipid portion” means (i) organic compounds that include fatty acid esters or derivatives thereof, are insoluble in water but soluble in many organic solvents, and include, but are not limited to, simple lipids such as fats, oils, and waxes, complex lipids such as phospholipids, glycolipids, cationic lipids, non-cationic lipids, neutral lipids, anionic lipids, and derivative lipids such as steroids; and (ii) organic compounds that do not include fatty acid esters but mimic such organic compounds by their amphiphilicity, that is, possessing both hydrophobic and hydrophilic portions, and therefore can aggregate in a specific manner in an aqueous environment to form layers, vesicles, and LNPs.

[0090] In this specification, "small interfering RNA (siRNA)" refers to a class of double-stranded RNA molecules, 16 to 40 nucleotides in length, that are involved in RNA interference (RNAi) pathways that interfere with the expression of specific genes. In addition to their role in RNAi pathways, siRNA also functions in RNAi-related pathways.

[0091] In this specification, “sgRNA” refers to a class of guide RNA molecules involved in CRISPR-Cas9 genome editing, in which sgRNA provides a precise genome editing template that minimizes “off-target” editing and maximizes “on-target” editing.

[0092] In this specification, "saRNA" refers to a class of RNA molecules that are "self-amplifying" or "self-replicating" due to their replicase properties, which result in multiple copies of the RNA.

[0093] In this specification, “RNA” means a molecule containing at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, auto-amplified RNA, sgRNA, gRNA, and multivalent RNA. “Ribonucleotide” means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribo-furanose moiety, and includes, but is not limited to, modified ribonucleotides. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinant-produced RNA, and modified RNA that differs from native RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides.

[0094] Cationic POZ Without being bound by any particular theory, cationic POZs appear to be a suitable substitute for ionizable lipids in stable LNPs. Therefore, without being bound by any particular theory, efficient and therapeutic delivery of RNA and DNA payloads is possible with LNPs constructed using cationic POZs (without using conventionally used ionizable lipids).

[0095] For example, it has now been discovered that cationic poly(ethyl oxazoline) (cationic PEOZ) yields stable LNPs that exhibit a specific dependence on flow rate ratio when incorporating oligonucleotides for therapeutic delivery. In this embodiment, LNPs prepared by microfluidic techniques produce large particles at very low flow rates and flow rate-independent limit-size LNPs at slightly higher flow rates. This highly efficient production of LNPs without any ionizable lipid components is not only remarkable and unexpected but can also have profound implications for a variety of RNA and DNA therapeutics, including vaccines, cancer immunotherapy, and gene therapy. In fact, the cationic POZ LNPs of this disclosure may have no immunogenicity or reduced immunogenicity compared to corresponding LNPs containing ionizable lipids and PEG-lipids, and thus can provide a safer method for delivering payloads.

[0096] In some embodiments, the cationic POZ can be a POZ-cation of formula I: R-POZ-cation (I) [In the formula, R includes an initiator group.] In some embodiments, R includes hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, or a substituted or unsubstituted aralkyl group. POZ can be a polyoxazoline polymer. In some embodiments, POZ can be poly(ethyloxazoline). In other embodiments, POZ has the structure [N(COR2)CH2CH2] n [In the structure, R2 is ethyl, and n ranges from 1 to 1000.] This can be a polyoxazoline polymer. The POZ cation may be any of the cationic POZs synthesized in the examples. For example, in some embodiments, the cationic POZ is PEOZ colamide.

[0097] In other embodiments, the cationic POZ can be the POZ-cation of formula II:

[0098] [ka] [In the formula, R includes an initiator group, n ranges from 1 to 10, R2 is independently selected for each repeating unit from an unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional group, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.] In some embodiments, R includes hydrogen, a substituted or unsubstituted alkyl, an alkyne-substituted alkyl, a triazole having a bonded carboxylic acid, or a substituted or unsubstituted aralkyl group.

[0099] T may be any nucleophile capable of terminating the POZ polymer living cationic polymerization. In one embodiment, T is a thioalkyl carboxylic acid, a thiocarboxylic acid ester, or a hydroxyl group. In other embodiments, T comprises ZBQ, where Z comprises S, O, or N, B is an optional linking group, and Q is a termination nucleophile or a termination moiety of a nucleophile. In some embodiments, Z is S. In other embodiments, Z is O. In yet another embodiment, Z is N.

[0100] In some embodiments, Q is inert (i.e., contains no functional groups). In other embodiments, Q contains a functional group. When Q contains a functional group, suitable functional groups include, but are not limited to, alkyne, alkene, amine, oxyamine, aldehyde, ketone, acetal, thiol, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (e.g., but not limited to N-hydroxysuccinimidyl (NHS) and 1-benzotriazine active ester), active carbonate, chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS). Further, when Q contains a functional group, the functional group can be chemically orthogonal to one or more or all of the other functional groups present in the conjugate. When Q is a non-reactive group, any non-reactive group can be used, including, but not limited to, unsubstituted alkyl and -C6H5.

[0101] Examples of B include, but are not limited to, an alkylene group. In one embodiment, B is -(CH2) y - where y is an integer selected from 1 to 16. In some embodiments, y can be an integer selected from 1 to 10, 1 to 8, 1 to 6, or 1 to 4. For example, in one embodiment, y is 2.

[0102] In other embodiments, the cationic POZ can be a POZ-cation of formula III:

[0103] [Chemical formula] [In the formula, R includes an initiator, n ranges from 1 to 10, R2 is independently selected for each repeating unit from an unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional group, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group.] In some embodiments, R includes hydrogen, a substituted or unsubstituted alkyl, an alkyne-substituted alkyl, a triazole having a bonded carboxylic acid, or a substituted or unsubstituted aralkyl group. T may be any of the terminal groups described above in relation to Formula II.

[0104] In other embodiments, the cationic POZ can be the POZ-cation of formula IV:

[0105] [ka] [In the formula, R includes a branched group, n ranges from 1 to 10, R2 is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups for each repeating unit, m ranges from 1 to 100, a ranges from 3 to 20, and T includes a terminal group.] In some embodiments, R can be glycerol, pentaerythritol, polyglycerol, etc. T may be any of the terminal groups described above in relation to formula II.

[0106] Non-restrictive examples of such structure IV having a glycerol central branching point are as follows:

[0107] [ka] This glycerol-derived cationic POZ can be synthesized via the following pathway.

[0108] [ka]

[0109] Sterol lipids As briefly stated above, the cationic POZ LNPs prepared in accordance with this disclosure contain sterol lipids for stability. A suitable sterol lipid for use in accordance with this disclosure is cholesterol. Another suitable sterol lipid for use in accordance with this disclosure is plant sterols.

[0110] Helper lipids Cationic POZ LNPs prepared in accordance with this disclosure contain helper lipids that provide structural support and promote endocytosis. Helper lipids refer to amphiphilic lipids having hydrophobic and polar head group portions that can spontaneously form bilayer vesicles in water, as exemplified by phospholipids, or stably incorporate into lipid bilayers, with the hydrophobic portion in contact with the inner hydrophobic region of the bilayer membrane and the polar head group portion oriented toward the outer polar surface of the membrane. Such helper lipids typically comprise one or two hydrophobic acyl hydrocarbon chains or steroid groups, and the polar head group may contain a chemically reactive group, such as an amine, acid, ester, aldehyde, or alcohol. Non-limiting examples suitable for use in accordance with this disclosure include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically about 14 to 22 carbon atoms long and have a variable degree 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).

[0111] Polymer lipids In some embodiments, the polymer lipid can be a POZ-lipid of formula V: R1-POZ-L-lipid (V) [In the formula, R1 comprises an initiator, POZ comprises poly(ethyl oxazoline), L comprises a physiologically degradable linking group, and the lipid comprises an uncharged lipid comprising at least one hydrophobic moiety.] In other embodiments, POZ is [N(COR2)CH2CH2] n [Here, R2 is ethyl.] is included. In yet another embodiment, R1 includes hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, a triazole having a bonded carboxylic acid, or a substituted or unsubstituted aralkyl group. In yet another embodiment, L includes ethers, esters, carboxylic acid esters, carbonate esters, carbamates, amines, amides, urethanes, disulfides, and combinations thereof. In yet another embodiment, the lipid includes two hydrophobic moieties. In yet another embodiment, the lipid includes phospholipids, glycerolipids, dialkylacetamides, or combinations thereof. In yet another embodiment, the lipid includes 1,2-dimiristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or combinations thereof.

[0112] In other embodiments, the polymer lipid may be a POZ-lipid of formula VI; Lipid-L1-(POZ) n a -T (VI) [In the formula, the lipid comprises an uncharged lipid containing at least one hydrophobic moiety, L1 comprises a physiologically degradable linking group, POZ comprises a polyoxazoline polymer of the structure [N(COR2)CH2CH2] [wherein R2 is ethyl], n ranges from 1 to 1,000, a is ran representing a random copolymer or block representing a block copolymer, and T comprises a terminal group.]

[0113] In some embodiments, L1 includes ethers, esters, carboxylic acid esters, carbonate esters, carbamates, amines, amides, urethanes, disulfides, and combinations thereof. In other embodiments, L1 includes triazoles.

[0114] In yet another embodiment, T comprises ZBQ, where Z comprises S, O, or N, B is an optional linking group, and Q is a termination nucleophile or a portion thereof. In yet another embodiment, the lipid comprises two hydrophobic moieties. In yet another embodiment, the lipid comprises phospholipids, glycerolipids, dialkylacetamides, or combinations thereof. In yet another embodiment, the lipid comprises 1,2-dimiristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or combinations thereof.

[0115] In yet another embodiment, the polymer lipid can be a POZ-lipid of formula VII: R1-(POZ) n a -Z-L2-lipid (VII) [In the formula, R1 contains an initiator group, POZ contains a polyoxazoline polymer of the structure [N(COR2)CH2CH2] [where R2 is ethyl], n ranges from 1 to 1,000, a is ran representing a random copolymer or block representing a block copolymer, Z contains S, O, or N, L2 contains a physiologically degradable linking group, and the lipid contains an uncharged lipid containing at least one hydrophobic group.]

[0116] In some embodiments, L2 includes ethers, esters, carboxylic acid esters, carbonate esters, carbamates, amines, amides, urethanes, disulfides, and combinations thereof. In other embodiments, the lipid includes two hydrophobic moieties. In yet another embodiment, the lipid includes phospholipids, glycerolipids, dialkylacetamides, or combinations thereof. In yet another embodiment, the lipid includes 1,2-dimiristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or combinations thereof. In yet another embodiment, R1 includes hydrogen, or substituted or unsubstituted alkyl, and n ranges from 15 to 35.

[0117] In yet another embodiment, the polymer lipid can be a POZ-lipid of formula VIII:

[0118] [ka] [In the formula, R1 comprises an initiator, L3 comprises a physiologically degradable linking group, the lipid comprises an uncharged lipid containing at least one hydrophobic moiety, n ranges from 1 to 5, R2 is independently selected for each repeating unit from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T comprises a terminal group.]

[0119] In some embodiments, L3 includes esters, carboxylic acid esters, carbonate esters, carbamates, amides, and combinations thereof. In other embodiments, L3 includes triazoles. In yet another embodiment, T includes ZBQ, where Z includes S, O, or N, B is an optional linking group, and Q is a termination nucleophile or a portion thereof. In yet another embodiment, the lipid includes two hydrophobic moieties. In yet another embodiment, the lipid includes phospholipids, glycerolipids, dialkylacetamides, or combinations thereof. In yet another embodiment, the lipid includes 1,2-dimiristoyl-sn-glycerol, 1,2-dilauroyl-sn-glycerol, or combinations thereof.

[0120] Further specific embodiments and examples of POZ lipids suitable for use as polymer lipids in cationic POZ LNPs prepared in accordance with this disclosure are described in U.S. Provisional Patent Application No. 63 / 440,210, the entire disclosure of which is incorporated herein by reference.

[0121] payload As briefly stated above, the cationic POZ LNP formed in accordance with this disclosure may also include a payload. In this embodiment, the payload may be an oligonucleotide, a protein, or a combination thereof.

[0122] In one embodiment, the oligonucleotide includes DNA, siRNA, self-replicating mRNA (saRNA), mRNA consisting of modified nucleosides, and mRNA consisting of native nucleosides. In one embodiment, the oligonucleotide is DNA. In another embodiment, the oligonucleotide is siRNA. In yet another embodiment, the oligonucleotide is saRNA, mRNA consisting of modified nucleosides, or mRNA consisting of native nucleosides. In yet another embodiment, the oligonucleotide is sgRNA used in genome editing.

[0123] Oligonucleotides can be encapsulated in LNPs with high efficiency. In one embodiment, oligonucleotides are encapsulated in LNPs with an efficiency of at least 90%. In another embodiment, oligonucleotides are encapsulated in LNPs with an efficiency of about 90 to about 99%. In yet another embodiment, oligonucleotides are encapsulated in LNPs with an efficiency of about 90 to about 95%. In yet another embodiment, oligonucleotides are encapsulated in LNPs with an efficiency of more than about 95%.

[0124] Similarly, polyplexes can be produced with high efficiency using oligonucleotides and POZ polymers. In some embodiments, oligonucleotides are incorporated into the polyplex with an efficiency of at least 90%, about 90–99%, about 90–95%, or more than 95%.

[0125] Characteristics of cationic POZ LNPs The particle sizes of cationic POZ LNPs prepared according to this disclosure can vary. In one embodiment, the cationic POZ LNPs formed according to this disclosure are amphiphilic spherical vesicles ranging in size from about 10 nm to about 10 microns, formed by one or more lipid bilayers surrounding an aqueous core. In another embodiment, the cationic POZ LNPs formed according to this disclosure have a particle size of about 25 nm to about 8 microns. In yet another embodiment, the cationic POZ LNPs formed according to this disclosure have a particle size of about 30 nm to about 5 microns. In this embodiment, the particle size of the cationic POZ LNPs can be about 20 nm to about 3 microns. In another embodiment, the cationic POZ 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. A method for size fractionation is disclosed herein. However, in some embodiments, size fractionation is not required.

[0126] Method for preparing cationic POZ LNPs and polyplexes The cationic POZ LNP compositions of this disclosure can be prepared by various methods. In one embodiment, liposomes are prepared by reverse-phase evaporation (Szoka et al., PNAS 1978, vol. 75, 4194-4198; Smirnov et al., Byulleten' Eksperimental'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 or may not contain linked target molecules, is mixed with a smaller volume of aqueous medium, and the mixture is dispersed to form a water-in-oil emulsion, preferably using a pyrogen-free component. The target molecule to be delivered is added to the lipid solution if it is a lipophilic target molecule, or to the aqueous medium if it is a water-soluble target molecule. The lipid solvent is evaporated and removed, 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 overwhelmingly oligolamellar, i.e., containing one or more lipid bilayer shells. REVs can be readily sized by extrusion to obtain oligolayer vesicles having a selected size, preferably about 0.05–0.2 microns, as described below.

[0127] Furthermore, multilayer vesicles (MLVs) can be created. In this method, a mixture of liposome-forming lipids, which may include polymer lipids having or not having linked target molecules as described herein, is dissolved in a suitable solvent, and the solvent is evaporated in a container to form a thin film. The thin film is then covered with an aqueous medium. The lipid film is hydrated to form an MLV. MLVs generally exhibit a size of about 0.1 to 10 microns. MLVs can be sized to a desired size range by extrusion and other methods described herein.

[0128] One effective sizing method for REV and MLV involves extruding an aqueous suspension of liposomes through a polycarbonate membrane having 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 membrane pore size generally corresponds to the maximum size of the liposomes produced by extrusion through the membrane, especially if the preparation has been extruded through the same membrane two or more times. A method for sizing larger MLVs is provided by Zhu et al. (PLoS One. 2009;4(4):e5009. Epub 2009 Apr 6).

[0129] When smaller particle sizes are desired, REV or MLV preparations can be processed to produce small monolayer vesicles (SUVs) characterized by sizes in the 0.04–0.08 micron range. Such particles may be useful in targeting tumor or lung tissue if the particles can be absorbed through capillary walls (particles larger than 0.1 microns may not be absorbed).

[0130] Cationic POZ LNPs may be treated to remove foreign components before use. For example, if surfactants are used as described above, excess surfactants may be removed before use. Furthermore, if payloads such as oligonucleotides as described above are trapped in the cationic POZ LNP composition, excess or untrapped payloads may be removed before use. Separation techniques for performing this task are known in the art, and the choice of a particular method may depend on the nature of the component to be removed. Preferred methods include, but are not limited to, centrifugation, dialysis, and molecular sieve chromatography. The composition may be sterilized by filtration through a standard 0.22 micron filter.

[0131] Cationic POZ LNPs can be prepared by a traditional method that involves hydrating a lipid film containing polymer lipids, cationic POZ, helper lipids, and cholesterol. This method involves dissolving these materials in an organic solvent such as chloroform or dichloromethane, then evaporating the solvent to produce a thin film. The thin film is then hydrated with an aqueous buffer containing a drug or nucleic acid to passively encapsulate the payload. LNPs, which are typically low-encapsulation heterogeneous particles, are formed and require size reduction by extrusion or sonication.

[0132] Another preferred technique involves rapid mixing using a microfluidic device. The lipid stock solution is prepared by dissolving lipids in an organic solvent such as ethanol. The aqueous stock solution contains nucleic acids dissolved in a buffer with known pH, ionic strength, and buffering capacity. The two stock solutions are passed through a micromixer at a predetermined rate to allow cationic lipids to interact with negatively charged nucleic acids, resulting in higher encapsulation efficiency (i.e., >90%) and a 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, centrifugation, or other techniques. LNPs of a specified size are generated by controlling microfluidic manipulation parameters, resulting in LNPs with low polydispersity and uniform particle size. The average particle diameter (<100 nm), polydispersity (<0.40 and, more specifically, <0.20), and zeta potential of the LNPs are three methods used to characterize the preparations.

[0133] To enable optimal size, high payload release and transfection, and improved stability of the hydrated formulation, the ratio of cationic POZ lipids to polymer lipids to cholesterol can be varied. In one embodiment, the mol% of cationic POZ in the LNP is about 30% to about 70%. In some embodiments, the mol% of cationic POZ in the LNP is about 40% to about 60%. In other embodiments, the mol% of cationic POZ in the LNP is about 45% to about 55%. In this regard, the remainder of the LNP can be about 30% to about 50% sterol lipids, about 0.5% to about 20% polymer lipids, and about 5% to about 15% helper lipids. In some embodiments, the mol% of sterol lipids in the LNP can range from about 35% to about 45%. In other embodiments, the mol% of polymer lipids can range from about 0.5% to about 10%, about 0.5% to about 5%, or about 1% to about 3%. In yet another embodiment, the mol% of the helper lipid can range from about 7% to about 12%, about 8% to about 11%, or about 6% to about 14%.

[0134] In yet another embodiment, the LNP may consist of about 0.1% to about 10% cationic POZ, about 30% to about 80% sterol lipids, about 0.5% to about 20% polymer lipids, and about 15% to about 65% helper lipids. In some embodiments, the mol% of cationic POZ in the LNP is about 0.2% to about 8%. In another embodiment, the mol% of cationic POZ in the LNP is about 0.3% to about 5%. In yet another embodiment, the mol% of sterol lipids in the LNP may range from about 35% to about 78%, about 30% to about 40%, about 70% to about 80%, or about 55% to about 65%. In yet another embodiment, the mol% of polymer lipids may range from about 0.5% to about 10%, about 0.5% to about 5%, or about 1% to about 3%. In yet another embodiment, the mol% of the helper lipid can range from about 18% to about 63%, about 17% to about 25%, about 30% to about 40%, or about 55% to about 65%.

[0135] Similarly, polyplexes can be prepared in accordance with this disclosure using cationic polymers and payloads disclosed herein. In some embodiments, a solution of the cationic polymer can be mixed with a payload solution.

[0136] Administration The cationic POZ LNPs of this disclosure can be delivered to any cell. After in vivo administration of the cationic POZ LNPs, the payload is released. In this embodiment, the cationic POZ LNPs of this disclosure can be incorporated into a pharmaceutical composition that can induce the treatment of a disorder or disease. For example, a pharmaceutical composition comprising cationic POZ LNPs prepared according to this disclosure can be used to prevent or treat infectious diseases, including but not limited to SARS-CoV-2, rabies, and influenza, by specifically targeting certain immune cells other than antigen-presenting cells. Furthermore, a pharmaceutical composition comprising cationic POZ LNPs prepared according to this disclosure can be used as a therapeutic agent for cancer and genetic diseases. Such a pharmaceutical composition may also include a pharmaceutically acceptable carrier in addition to the cationic POZ LNPs.

[0137] In one embodiment, a pharmaceutical composition containing an effective amount of the cationic POZ LNP of the Disclosure may be delivered to an animal. In this embodiment, the delivery of an effective amount of the cationic POZ LNP of the Disclosure may be via subcutaneous, intravenous, intramuscular, intradermal, or aerosol pathways. In one embodiment, the animal is a human. [Examples]

[0138] The following examples are not intended to limit the present invention or the claimed subject matter. Rather, they are intended to further illustrate embodiments of the present disclosure.

[0139] Example 1. Synthesis of H-PEOZ-NHS ester 2K

[0140] [ka]

[0141] 5.00 g of dipotassium peozoate (MW 2271.9 Da, 2.201 mmol, 1.00 equivalent) was added to a 250 mL round-bottom flask, followed by 50 mL of acetonitrile. The solution was evaporated to dryness by rotational evaporation. The residual white solid was dissolved in 50 mL of dimethyl chloride (DCM). Next, N-hydroxysuccinimide (284.3 mg, 2.421 mmol, 1.10 equivalents) was added to the clarified solution under an argon atmosphere. DCC (490.8 mg, 2.355 mmol, 1.07 equivalents) was added all at once, and the resulting solution was stirred overnight at room temperature. The turbid reaction mixture was filtered through sintered glass frit. The resulting filtrate was concentrated to dryness by rotational evaporation. The remaining solid was redissolved in 27 mL of DCM, and then slowly transferred to a beaker containing a 500 mL stirred MTBE solution. The precipitate was collected via vacuum filtration, and the solid was then transferred to a 100 mL round-bottom flask and dissolved in methanol (15 mL). The solution was evaporated to dryness by rotary evaporation, and the remaining solid was vacuum-dried to obtain 4.2 g of a white solid.

[0142] 1 1HNMR analysis showed standard signals for PEOZ-NHS ester 2K (500 MHz, DMSO-d6): δ 3.32 (CH2CH2 skeleton); 2.34 (C(O)-CH2); 0.97 (CH3). An additional signal for the terminal NHS ester was present at δ 2.81 (COCH2CH2CO).

[0143] Example 2. (EOZ) 12 Synthesis of (PrAcidOZ)8-OH 2K and POZ-OH 2K 8p acids

[0144] [ka]

[0145] A 50 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet is subjected to MeEstOZ under an argon atmosphere. * (2.12 g, 13.5 mmol, 8 equivalents) and 2-ethyl-2-oxazoline (2.00 g, 20.2 mmol, 12 equivalents) were added, followed by the addition of a stirring bar and PhCl (17 mL). TfOH (148.8 μL, 1.68 mmol, 1.00 equivalent) was added dropwise, and the mixture was stirred at room temperature for 5 minutes. The reaction product was then heated to 80°C and stirred for 120 minutes. The polymerization mixture was then cooled to room temperature, and an aqueous solution of Na2CO3 (0.712 g, 6.72 mmol, 4.00 equivalents) in H2O (17 mL) was added. The reaction mixture was stirred for at least 12 hours. Following this time, PhCl was removed using a rotary evaporator. 1N NaOH (aqueous solution) (6 mL) was added to the resulting aqueous solution, and the mixture was stirred overnight. The mixture was acidified with 3N HCl (aqueous solution), and NaCl (6.5 g, 15 w / v%) was added. DCM (25 mL) was added to the aqueous mixture while stirring, and a white, sticky mass of precipitate formed. The entire solution was decanted, and the residue was washed by swirling and decanting with DCM (25 mL). The residue was dried under reduced pressure to obtain 2.6 g (65% yield) of the desired product as a white powder.

[0146] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6, δ) analysis showed standard skeleton signals for PEOZ at 7.82 (terminal NH), 3.34 (CH2CH2 skeleton), 3.16 (N-CH2), 2.32-2.27 (C(O)-CH2-), and 0.94 (-CH3). Signals for pendant groups were present at 2.51-2.40 ppm (-CH2CH2CO2H pendent). The number of pendant groups was calculated in advance from the polymer starting material to be 7.19. MALDI analysis showed Mn 2520Da and PDI 1.105.

[0147] *MeEstOZ was synthesized using a modified literature procedure: Bouten, PJM; Hertsen, D.; Vergaelen, M.; Monnery, BD; Boerman, MA; Goossens, H.; Catak, S.; van Hest, JCM; van Speybroeck, V.; Hoogenboom, R., Polym. Chem., 2015, 6, pp. 514-518.

[0148] Example 3. Synthesis of (EOZ)5(PrAcidOZ)4-Pip-COOH 1.3K

[0149] [ka]

[0150] PEOZ-PipEt ester 1.2K 4P Me ester. In a 50 mL oven-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, MeEstOZ (1.466 g, 11.5 mmol, 4 equivalents) and 2-ethyl-2-oxazoline (1.429 g, 14.4 mmol, 5 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and PhCl (13 mL). TfOH (255.1 μL, 2.88 mmol, 1.00 equivalent) was added dropwise, and the mixture was stirred at room temperature for 5 minutes. The reaction product was then heated to 110 °C and stirred for 40 minutes. The polymerization mixture was then cooled to room temperature, and ethyl isonipecoate (0.91 mL, 5.77 mmol, 2.00 equivalents) was added. The reaction mixture was stirred at room temperature for at least 12 hours. Following this time, all volatile substances were removed using a rotary evaporator, and then azeotropic analysis was performed using a 0.5N HCl (aqueous solution) (15 mL). The resulting aqueous solution was passed through an Amberlite column (IR120H / IRA67), and the recovered filtrate was freeze-dried using a freeze-dryer to obtain 3.61 g (97.7% yield) of the desired product as white crystals.

[0151] 1¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed standard skeleton signals for PEOZ at 7.82 (terminal NH), 4.08 (m, terminal piperidine C(=O)OCH2CH3, 2H), 3.64 (m, piperidine CH, 1H), 3.36 (CH2CH2 skeleton), 3.15 (N-CH2), 2.58 (m, terminal piperidine -NCH2-, 4H), 2.31 (C(O)-CH2-), 1.84 (m, terminal piperidine -NCH2CH2, 4H), 1.19 (m, terminal piperidine -OCH2CH3), 0.95 (-CH3). Signals for the pendant group were present at 2.96 and 2.58 ppm (pendent's -CH2CH2CO2CH3) and 3.55 ppm (s, pendent's -OCH3). The ratio of EOZ:MeEstOZ:terminal ester was determined to be 5:4.4:0.9. MALDI analysis showed Mn 1239Da and PDI 1.08.

[0152] PEOZ-Pip-acid 1.2K 4P acid. The PEOZ-PipEt ester 1.2K 4P Me ester recovered above was dissolved in 1.0N NaOH (aqueous solution) (28.8 mL) to obtain a pH 13 solution. The reaction mixture was stirred at room temperature for 18 hours, where the mixture was acidified to pH=3. The obtained solution was washed with DCM (20 mL). The aqueous layer was collected and concentrated using a rotary evaporator to obtain a concentrated slush. The residue was dissolved in DMF (14 mL), filtered using a syringe filter, and concentrated using a rotary evaporator. The resulting sticky gel was stirred in THF, sonicated, and decanted. This process was repeated in diethyl ether. The residue was dried in complete vacuum to obtain 3.47 g of the target product as white crystals.

[0153] 1¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed standard skeleton signals of PEOZ at 7.82 (terminal NH), 3.36 (CH2CH2 skeleton), 3.15 (N-CH2), 2.05 (m, terminal piperidine -NCH2-, 4H), 2.28 (C(O)-CH2-), 1.75 (m, terminal piperidine -NCH2CH2), and 0.96 (-CH3). Signals for the pendant group were present at 2.53 and 2.41 ppm (pendent's -CH2CH2CO2H). Completion of hydrolysis was confirmed by the disappearance of peaks for the ester group at 3.55 ppm (-OCH3), 4.08 (m, C(=O)OCH2CH3), and 1.19 (m, -OCH2CH3).

[0154] Example 4. Synthesis of PEOZ-propargylamide 2K

[0155] [ka]

[0156] In a 100 mL round-bottom flask equipped with a stirring bar, PEOZ-NHS ester 2K (4.00 g, MW 2369, 1.69 mmol, 1.00 equivalent) was dissolved in DCM (40 mL) under an argon atmosphere. Next, propargylamine (0.22 mL, 3.38 mmol, 2.00 equivalent) was added to the clarified solution, followed by triethylamine (0.47 mL, 3.38 equivalents, 2.00 equivalent). The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered through crude sintered glass frit. The resulting filtrate was concentrated to dryness by rotary evaporation. The remaining solid was redissolved in DCM (21 mL) and then slowly transferred to a beaker containing MTBE stirred solution (150 mL). The precipitate was collected by vacuum filtration, and the remaining solid was vacuum dried to obtain 3.9 g of a white solid.

[0157] 1HNMR analysis showed standard signals for PEOZ-propargylamide 2K (500 MHz, DMSO-d6): δ 3.32 (CH2CH2 skeleton); 2.34 (C(O)-CH2); 0.97 (CH3). Additional signals for the terminal propargylamide were present at δ 3.85 (N-CH2) and 3.75 (C≡CH).

[0158] Example 5. P[(EOZ) m (PtynOZ) n Synthesis of ]-propargylamide 2K Step 1. Synthesis of NHS esters [ka]

[0159] In a 250 mL round-bottom flask containing a stirring bar, P[(EOZ) 13 (PtynOZ)7]-acid 2K (MW 2356Da, 10.0 g, 4.24 mmol, 1.00 equivalent) was added, followed by DCM (100 mL). Next, N-hydroxysuccinimide (0.82 g, 4.67 mmol, 1.10 equivalent) was added to the clarified solution under an argon atmosphere. DCC (0.55 g, 4.54 mmol, 1.07 equivalent) was added all at once, and the resulting solution was stirred overnight at room temperature. The reaction mixture was filtered through sintered glass frit. The obtained filtrate was concentrated to dryness by rotary evaporation. The remaining solid was redissolved in DCM (55 mL) and then slowly transferred to a beaker containing MTBE stirred solution (400 mL). The precipitate was recovered by vacuum filtration, and the remaining solid was vacuum dried to obtain 10.4 g of white solid.

[0160] 1 HNMR analysis is P[(EOZ)] 13(PtynOZ)7]-NHS ester 2K's standard signal was shown (500 MHz, DMSO-d6): δ 3.32 (CH2CH2 skeleton); 2.34 (C(O)-CH2); 1.66 (pendent alkyne-CH2-); 0.97 (CH3). An additional signal for the terminal NHS ester was present at δ 2.81 (COCH2CH2CO).

[0161] Step 2. P[(EOZ) 13 Synthesis of (PtynOZ)7]-propargylamide 2K

[0162]

Chemical formula

[0163] In a 250 mL round-bottom flask equipped with a stir bar, (PtynOZ)7]-NHS ester 2K (10.4 g, 4.24 mmol, 1.00 equivalent) was dissolved in DCM (100 mL) under an argon atmosphere. Then, propargylamine (0.54 mL, 8.49 mmol, 2.00 equivalents) was added to the clear solution, and then triethylamine (1.18 mL, 8.49 equivalents, 2.00 equivalents) was added, and the reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered through a coarse sintered glass frit. The resulting filtrate was concentrated to dryness by rotary evaporation. The remaining solid was redissolved in DCM (55 mL) and then slowly transferred to a beaker containing a stirred MTBE solution (400 mL). The precipitate was collected by vacuum filtration, and the remaining solid was dried in vacuo to obtain 9.5 g of a white solid. <00​​​​​​​​The standard signals of propargylamide 2K were shown (500 MHz, DMSO-d6): δ 3.32 (CH2CH2 backbone); 2.34 (C(O)-CH2); 1.66 (alkyne-CH2CH2); 0.97 (CH3). Additional signals for the terminal propargylamide were present at δ 3.85 (N-CH2) and 3.75 (C≡CH).

[0165] Example 6. Synthesis of PEOZ-N,N-dimethylaminoethylamide 1K (PEOZ DMAEA 1K)

[0166] [Chemical formula]

[0167] PEOZ-NHS ester 1K (1.130 g, 1.004 mmol, 1.0 equivalent) in a 50 mL RB flask was dissolved in anhydrous DCM (20 mL). Under Ar, N,N-dimethylethylenediamine (121.5 μL, 1.090 mmol, 1.05 equivalents) was added to the clear solution. The clear solution was stirred overnight at room temperature. The solution was evaporated to dryness. The residual solid was dissolved in water (50 mL). The turbid solution was filtered. The filtrate was loaded onto an Amberlite IRA-67 column (5 g m) and then eluted with water (75 mL). The eluate was collected. The pH of the solution was adjusted to 12.40 with 1N NaOH. NaCl (18.8 g) was dissolved in the solution. The solution was extracted with DCM (3 × 75 mL). The DCM phase was dehydrated with anhydrous magnesium sulfate (1.6 g) and anhydrous sodium sulfate (84 g) for 1 hour. The mixture was filtered and the filtrate was evaporated to dryness. The residue was dried under vacuum overnight to obtain 945 mg of a white solid. The solid was dissolved in water (13.5 mL). The pH of the solution was adjusted from 9.80 to 6.75 with 1N HCl. The solution was then lyophilized to obtain 0.94 g of a white solid.

[0168] 1HNMR analysis showed standard signals for PEOZ DMAEA 1K (500 MHz, DMSO-d6): δ 3.32 (CH2CH2 skeleton); 2.31 (C(O)-CH2); 0.97 (CH3). An additional signal was observed for the terminal DMAEA group, δ 9.84 (N). + It was present in H), δ 8.18 (CO-NH-), and δ 2.70 (N-(CH3)2).

[0169] Example 7. Synthesis of PEOZ-Colamide 1K

[0170] [ka]

[0171] PEOZ-NHS ester 1K (0.768 g, 0.706 mmol, 1.0 equivalent) in a 100 mL RB flask was dissolved in anhydrous DMF (20 mL). Under an Ar atmosphere, coramine chloride hydrochloride (0.136 g, 0.776 mmol, 1.1 equivalents) was added to the clarified solution, followed by the addition of TEA (0.413 mL, 2.260 mmol, 4.2 equivalents). The clarified solution was stirred overnight at room temperature. After the overnight reaction, the solution was evaporated to dryness by rotary evaporation. The residual solid was dissolved in 2 mM HCl (17 mL). The turbid solution was filtered through a 0.2 μm GHP syringe filter, and the clarified filtrate was purified by reverse-phase chromatography using a Biotage SNAP Ultra C18 30 g column. The column was eluted using a gradient with 2 mM HCl and methanol. Elution was monitored using a UV detector. The desired fraction containing the product was collected and evaporated to dryness by rotational evaporation. The remaining solid was dissolved in water (20 mL) and passed twice through a column packed with Amberlite IRA-67 medium (5 g m). The column was then eluted with water. The collected eluent (50 mL) was then freeze-dried to obtain 0.64 g of yellow solid.

[0172] 1HNMR analysis showed standard signals for PEOZ colamide 1K (500 MHz, DMSO-d6): δ 3.32 (CH2CH2 skeleton); 2.31 (C(O)-CH2); 0.97 (CH3). Additional signals were observed for the terminal colamide groups: δ 8.41 (CO-NH-) and δ 3.10 (N). + It was located in -(CH3)3).

[0173] Example 8. Synthesis of PEOZ 2K colamide

[0174] [ka]

[0175] PEOZ-NHS (200.0 mg, 0.10 mmol, 2 kDa, 1.0 equivalent) and (2-aminoethyl)trimethylammonium chloride-HCl (21.2 mg, 0.120 mmol, 1.2 equivalents) were added to two drum vials. 0.1 N boric acid (2 mL) was added, and the mixture was stirred to completely dissolve it. The pH of the solution was adjusted to 8.5 by adding 0.1 N NaOH (aqueous solution) dropwise while stirring. After stirring overnight, the resulting mixture (pH 8.42) was passed through an amberlite column (IR120H / IRA67) and a DEAE column. After adjusting the pH to 5.0, the filtrate was freeze-dried using a freeze-dryer to obtain 74.0 mg (yield 37%) of the desired product as white crystals.

[0176] 1 ¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed standard skeleton signals for PEOZ at 7.82 (terminal NH), 3.34 (CH2CH2 skeleton), 3.16 (N-CH2), 3.02 (-S-CH2), 2.32-2.27 (C(O)-CH2-), and 0.94 (-CH3). An additional signal for the colamide moiety was observed at 3.09 (s, -CH2N). +It was present at (CH3)3, 9H, 2.73 (m, -NHCH2CH2NMe3, 2H), 2.40 (t, J = 7.0 Hz, -NHCH2CH2NMe3, 2H).

[0177] Example 9. (EOZ) 12 (Cholamide OZ) 6.5 Synthesis of -OH 2K, PEOZ-OH 2K 5.8P cholamide

[0178]

Chemical formula

[0179] PEOZ-OH 6.5P NHS. PEOZ-OH 6.5P acid (0.499 g, 2460 Da, 0.203 mmol, 1.0 equivalent) was added to a 25 mL flask and dried by azeotropy using DMF (0.5 mL) and MeCN (10 mL). The residue was dissolved in 1.5 mL of DMF and then diluted with 9 mL of DCM to obtain a slightly turbid solution. NHS (0.172 g, 1.462 mmol, 7.2 equivalents) and DCC (0.305 g, 1.462 mmol, 7.2 equivalents) were added to the solution, and then stirred for 18 hours. The reaction mixture was filtered using a syringe filter, and the filtrate was concentrated using a rotary evaporator to obtain a thick oily crude product. The crude product was dissolved in DCM and precipitated by adding it to diethyl ether with stirring. The precipitate was filtered, collected, and dried under reduced pressure to obtain 0.63 g of the desired product as a white powder.

[0180] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6,δ) Analysis showed standard skeletal signals of PEOZ at 7.82 (terminal NH), 3.34 (CH2CH2 skeleton), 3.16 (N-CH2), 2.32-2.27 (C(O)-CH2-), and 0.94 (-CH3). The pendant group signal was present at 2.88-2.55 ppm (-CH2CH2CO2H pendent) and shifted down to 2.51-2.40 ppm due to NHS ester formation. An additional signal for the NHS moiety was present at 2.78 ppm (-CH2CH2-NHS ring). The number of pendant groups was: 1 The value was determined to be 6.5 based on 1H NMR analysis.

[0181] PEOZ-OH 2K 5.8P coramide (calculated Mn 3370 Da). PEOZ-OH 2K 6.5P NHS (200.0 mg, 0.0646 mmol, 3100 kDa, 1.0 equivalent) and (2-aminoethyl)trimethylammonium chloride-HCl (85.0 mg, 0.485 mmol, 7.2 equivalents) were added to a 20 mL vial. 0.1 N boric acid (4 mL) was added, and the mixture was stirred to completely dissolve it. The pH of the solution was adjusted to 8.5 by adding 0.1 N NaOH (aqueous solution) dropwise while stirring. After stirring overnight, the resulting mixture (pH 8.39) was passed through an amberlite column (IR120H / IRA67). The filtrate was freeze-dried using a freeze-dryer to obtain 156.4 mg (71.8% yield) of the desired product as white crystals.

[0182] 1 ¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed standard skeleton signals for PEOZ at 3.30 (CH2CH2 skeleton), 2.32-2.27 (C(O)-CH2-), and 0.95 (-CH3). An additional signal for the colamide moiety was observed at 3.10 (s, -CH2N). + It was present at (CH3)3, 9H), 3.47(m, -NHCH2CH2NMe3, 2H), and 2.40(t, J=7.0Hz, -NHCH2CH2NMe3, 2H). The number of pendant colamide groups was 1The value was determined to be 5.8 based on 1H NMR analysis.

[0183] Example 10. Synthesis of (EOZ)5(CholineEsterOZ)5-Pip-CholineEster

[0184] [ka]

[0185] In two drum vials, under an argon atmosphere, (EOZ)5(PrAcidOZ)5-Pip-COOH 1.3K (200.0 mg, 0.149 mmol, 1340 Da, 1.0 equivalent), choline chloride (145.9 mg, 1.045 mmol, 7.0 equivalents), and DMAP (18.6 mg, 0.152 mmol, 0.1 equivalent) were added, followed by the addition of a stirring bar and DMF (3 mL). After adding DIC (0.162 mL, 1.045 mmol, 7 equivalents), the mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered using a syringe filter, and the filtrate was concentrated to approximately 1.5 mL. Precipitation was then performed using 18 mL of diethyl ether. The ether solution was decanted, and the residue was stirred with freshly added diethyl ether (15 mL). The white precipitate was filtered, collected, and dried in a vacuum to obtain 208 mg (67% yield) of the desired product as white crystals.

[0186] 1 ¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed the standard skeleton signals of PEOZ at 3.34 (CH2CH2 skeleton), 3.15 (N-CH2), 2.31 (-C(O)-CH2CH3), 2.05-1.53 ​​(m, terminal piperidine -CH2CH2-), and 0.96 (-C(O)CH2CH3). The signal of the pendent-type terminal choline moiety was 4.44 (br s, -OCH2CH2N + (CH3)3), 3.68(br s, -OCH2CH2N + (CH3)3), 3.15(br s, -OCH2CH2N +(CH3)3), 2.71 and 2.53 (pendent -CH2CH2C(O)O-). The number of pendant-type choline groups was 1 The value was determined to be 4.8 based on 1H NMR analysis.

[0187] Example 11. (EOZ)5(MePipEsterOZ) n - Synthesis of Pip-MePipEster1.2K

[0188] [ka]

[0189] In two drum vials, (EOZ)5(PrAcidOZ)4-Pip-COOH 1.2K, N-methyl-4-piperidinol, and DMAP were added under an argon atmosphere, followed by the addition of a stirring bar and DMF. After adding DCC, the mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered using a syringe filter, and the filtrate was concentrated. The residue was stirred with diethyl ether, and then the solution was decanted. After drying in a vacuum, the obtained material was dissolved in DCM and precipitated by adding it to diethyl ether with stirring. The white precipitate was filtered, collected, and dried in a vacuum to obtain the desired product as white crystals.

[0190] PEOZ-Pip 1.3K 1P MePipEster was synthesized using (EOZ)5(PrAcidOZ)4-Pip-COOH 1.2K (0.300g, 0.212mmol, 84.7 wt%, 1.0 equivalent), N-methyl-4-piperidinol (0.035g, 0.300mmol, 1.42 equivalents), DMAP (0.0061g, 0.050mmol, 0.24 equivalents), DCC (0.0625g, 0.300mmol, 1.42 equivalents), and DMF (3mL), yielding 0.254g (91% yield) of the desired product.

[0191] 1¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed the standard skeleton signals of PEOZ at 3.34 (CH2CH2 skeleton), 3.15 (N-CH2), 2.31 (-C(O)-CH2CH3), 2.05-1.22 (m, -CH2CH2- of terminal piperidine), and 0.94 (-C(O)CH2CH3). Signals for pendent-type terminal methylpiperidine moieties were present at 4.63 (br s, -OCH< of MePip), 2.03 (>NCH3 of MePip), 2.84, and 2.49 (-CH2CH2C(O)O- of pendent). The number of pendent-type N-methylpiperidine groups was: 1 The value was determined to be 1.0 based on 1H NMR analysis.

[0192] PEOZ-Pip 1.3K 5P MePipEster was synthesized using (EOZ)5(PrAcidOZ)4-Pip-COOH 1.2K (0.312g, 0.220mmol, 84.7 wt%, 1.0 equivalent), N-methyl-4-piperidinol (0.168g, 1.431mmol, 6.5 equivalents), DMAP (0.0054g, 0.044mmol, 0.2 equivalents), DCC (0.298g, 1.431mmol, 6.5 equivalents), and DMF (3mL), yielding 0.228g (61% yield) of the desired product.

[0193] 1 ¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed the standard skeleton signals of PEOZ at 3.34 (CH2CH2 skeleton), 3.15 (N-CH2), 2.31 (-C(O)-CH2CH3), 2.05-1.22 (m, -CH2CH2- of terminal piperidine), and 0.94 (-C(O)CH2CH3). The signal for the pendented terminal methylpiperidine moiety was present at 4.75 (br s, -OCH< of MePip). The number of pendented N-methylpiperidine groups was: 1 The value was determined to be 3.0 based on 1H NMR analysis.

[0194] Example 12. Synthesis of (N-methyl-4-piperidyl)-3-azidopropionate

[0195] [ka]

[0196] To a solution of N-methyl-4-piperidinol (0.14 g, 1.19 mmol, 1 equivalent) in DCM (8 mL), 3-azidopropionyl chloride (0.330 g, 2.38 mmol, 96.5 wt%, 2 equivalents) was slowly added at room temperature. After stirring for 18 hours, the reaction mixture was quenched with aqueous NaHCO3, extracted with additional DCM, and the organic layer was recovered. The mixture was dehydrated with Na2SO4, filtered, and concentrated under reduced pressure. Another purification was performed by flash chromatography (DCM / MeOH, 9 / 1) to obtain (N-methyl-4-piperidinol)-3-azidopropionate.

[0197] 1 ¹H NMR (Varian, 500 MHz, 10 mg / mL CDCl3, δ) analysis showed signals at 5.00 (br s, piperidine -OCH<, 1H), 3.55 (t, N3CH2CH2-, 2H), 2.96 (m, piperidine CH3NCH2CH2CHO-, 4H), 2.60 (t, N3CH2CH2-, 2H), 2.60 (s, CH3-piperidine, 3H), 2.14 (m, piperidine CH3NCH2CH2CHO-, 2H), and 1.95 (m, piperidine CH3NCH2CH2CHO-, 2H).

[0198] Example 13. Synthesis of (3-azidopropyl)N-methylpiperidinoate

[0199] [ka]

[0200] In a 100 mL round-bottom flask, 3-azido-1-propanol (1.49 g, 14.2 mmol, 96+%, 1.0 equivalent), 1-methylpiperidine-4-carboxylic acid HCl (2.80 g, 15.6 mmol, 1.0 equivalent), anhydrous DCM (56 mL), and DMAP (0.173 g, 0.1 equivalent, 1.42 mmol) were added. DCC (3.25 g, 1.1 equivalent, 15.6 mmol, 99%) was added to the solution, and the resulting solution was stirred at room temperature for 18 hours. The reaction mixture was filtered through a medium-grade sintered glass frit, and the frit was then rinsed with additional DCM. The resulting solution was stirred with saturated NaHCO3 (25 mL) for 5 minutes. The organic phase was collected, dehydrated with Na2SO4, filtered, and concentrated using a rotary evaporator. The residue was purified by silica gel column chromatography using Biotage and a mixture of DCM and methanol as the eluent to obtain the desired product as a clear, pale yellow oil (2.95 g, 92% yield).

[0201] 1 ¹H NMR (Varian, 500 MHz, 10 mg / mL CDCl3, δ) analysis showed signals at 4.17 (t, J = 6.0 Hz, N3CH2CH2CH2O-, 2H), 3.38 (t, J = 6.5 Hz, N3CH2-, 2H), 2.81 (br d, J = 11.0 Hz, Pip(-CHH)2CH-C(=O)O-, 2H), 2.28 (m, Pip(-CH2)2CH-C(=O)O-, 1H), 2.26 (s, Pip N-CH3, 3H), 1.98 (t, J = 11.3 Hz, Pip(-CHH)2CH-C(=O)O-, 2H), 1.92 (t, J = 6.5 Hz, N3CH2CH2-, 2H), 1.91 (t, J = 6.3 Hz, Pip(-CHH)2N-CH3, 2H), 1.78 (m, Pip(-CHH)2N-CH3, 2H).

[0202] Example 14. Synthesis of (choline iodide)-3-azidopropionate Step 1: Synthesis of (2-dimethylamino)ethyl 3-azidopropionate

[0203] [ka]

[0204] In a 250 mL round-bottom flask, 2-dimethylaminoethanol (5.70 mL, 1.0 equivalent, 56.0 mmol, >99.5%), 3-azidopropionic acid (6.38 mL, 1.2 equivalents, 67.2 mmol, 97% by weight), anhydrous toluene (150 mL), and DMAP (0.68 g, 0.1 equivalent, 5.60 mmol) were added. DCC (14.0 g, 1.2 equivalents, 67.2 mmol, 99%) was added to the solution, and the resulting solution was stirred at room temperature for 18 hours. The reaction mixture was filtered through a medium-weight sintered glass frit, and the frit was then rinsed with additional toluene. The resulting solution was concentrated using a rotary evaporator, and the residue was purified by silica gel column chromatography using biotage and a mixture of ethyl acetate and methanol as the eluent to obtain the desired product as a clear yellow oil (4.68 g, yield 45%).

[0205] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ): 4.13 (t, 2H, J = 6.00 Hz, -OCH2CH2N(CH3)2), 3.53 (t, 2H, J = 6.25 Hz, N3CH2CH2(C(=O)O-), 2.59 (t, 2H, J = 6.25 Hz, N3CH2CH2(C(=O)O-), 2.46 (t, 2H, J = 6.00 Hz, -OCH2CH2N(CH3)2), 2.15 (s, 6H, -N(CH3)2).

[0206] Step 2: Synthesis of (choline iodide)-3-azidopropionate

[0207] [ka]

[0208] To a solution of (2-dimethylamino)ethyl 3-azidopropionate (5.15 g, 27.7 mmol, 1.0 equivalent) in anhydrous DCM (40 mL), methyl iodide (1.74 mL, 27.7 mmol, 1.0 equivalent) was slowly added. After stirring for 18 hours, the reaction mixture (with a pale yellow precipitate) was slowly poured into diethyl ether (500 mL) with vigorous stirring. The precipitate was isolated by filtration using a moderate sintered glass frit and then rinsed with 200 mL of diethyl ether. The filtered white precipitate was transferred to a 50 mL bottle and dried under vacuum to obtain the desired product (8.39 g, yield 92.4%) as a pale yellow powder.

[0209] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ): 4.48 (t, 2H, J = 2.00 Hz, -OCH2CH2N + (CH3)3), 3.65 (t, 2H, J = 2.25 Hz, -OCH2CH2N + (CH3)3), 3.58 (t, 2H, J = 6.25 Hz, N3CH2CH2(C(=O)O-), 3.11 (s, 9H, -N + (CH3)3), 2.65 (t, 2H, J = 6.25 Hz, N3CH2CH2(C(=O)O-).

[0210] Example 15. Synthesis of (choline iodide)-2-azidopropionate Step 1: Synthesis of (2-dimethylamino)ethyl 2-azidopropionate

[0211] [ka]

[0212] In a 250 mL round-bottom flask, 2-dimethylaminoethanol (1.13 mL, 1.0 equivalent, 11.2 mmol, >99.5%), 2-azidopropionic acid (1.937 g, 1.5 equivalent, 16.8 mmol, 97% by weight), anhydrous DCM (25 mL), and DMAP (0.137 g, 0.1 equivalent, 1.12 mmol) were added. DIC (2.63 mL, 1.5 equivalent, 16.8 mmol, 99%) was added to the solution, and the resulting solution was stirred at room temperature for 18 hours. The reaction mixture was filtered through a medium-weight sintered glass frit, and the frit was rinsed with additional toluene. The resulting solution was concentrated using a rotary evaporator, and the residue was purified by silica gel column chromatography using biotage and a mixture of ethyl acetate and methanol as the eluent to obtain the desired product as a clear yellow oil (1.34 g, yield 64%).

[0213] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ): 4.29 (q, 1H, J = 7.0 Hz, N3CH(CH3) (C(=O)O-), 4.20 (m, 2H, -OCH2CH2N(CH3)2), 2.50 (t, 2H, J = 5.5 Hz, -OCH2CH2N(CH3)2), 2.16 (s, 6H, -N(CH3)2), 1.33 (d, 3H, J = 7.0 Hz, -N3CH(CH3)(C(=O)O-).

[0214] Step 2: Synthesis of (choline iodide)-3-azidopropionate

[0215] [ka]

[0216] To a solution of (2-dimethylamino)ethyl 2-azidopropionate (1.30 g, 6.96 mmol, 1.0 equivalent) in anhydrous DCM (13 mL), methyl iodide (0.44 mL, 6.96 mmol, 1.0 equivalent) was slowly added. After stirring for 18 hours, the reaction mixture (with a pale yellow precipitate) was slowly poured into diethyl ether (500 mL) with vigorous stirring. The precipitate was isolated by filtration using a moderate sintered glass frit and then rinsed with 200 mL of diethyl ether. The filtered white precipitate was transferred to a 20 mL vial and dried under vacuum to obtain the desired product (1.76 g, yield 77.3%) as a pale yellow powder.

[0217] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ): 4.55 (m, 2H, -OCH2CH2N + (CH3)3), 4.38 (q, 1H, J = 7.0 Hz, N3CH(CH3)(C(=O)O-), 3.69 (m, 2H, -OCH2CH2N + (CH3)3), 3.12 (s, 9H, -N + (CH3)3), 1.37 (d, 3H, J = 7.0 Hz, N3CH(CH3)(C(=O)O-).

[0218] Example 16. (PEOZ) 13 (PtynOZ) 9.5 Synthesis of -OH 2.6K (also known as PEOZ-OH 2.6K 9.5p)

[0219] [ka]

[0220] In a 250 mL oven-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, 15.680 g, 158.2 mmol, 14 equivalents of ethyl-2-oxazoline and 15.50 g, 113.0 mmol, 10 equivalents of pentynyl-2-oxazoline were added under an argon atmosphere. A stirring bar and 135 mL of PhCl were then added. 1.00 mL of TfOH (11.298 mmol, 1.00 equivalent) was added dropwise, and the mixture was stirred at room temperature for 5 minutes. The reaction product was then heated to 110°C over 20 minutes and stirred at that temperature for 40 minutes. After cooling to room temperature, the polymerization mixture was transferred to an oven-dried 1000 mL round-bottom flask and concentrated using a rotary evaporator to obtain a sticky residue. After adding an aqueous solution of Na2CO3 (452 ​​mL, 0.2 M, 2.00 equivalents), residual chlorobenzene was azeotropically evaporated (approximately 100 mL in total). The resulting aqueous solution was diluted with MeCN (250 mL) and stirred overnight. After removing some of the MeCN and water using a rotary evaporator, NaCl (30.4 g, 10% w / w) was added to the resulting aqueous solution, and the product was then extracted into DCM (2 times × 300 mL). The combined organic phase was dehydrated with Na2SO4, filtered, concentrated, and further dried in vacuum at 50°C to obtain the desired product (27.4 g, 92% yield) as white crystals.

[0221] 1¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed a normal skeleton peak at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.32 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.96 (m, 3H, -C(=O)CH2CH3). The pendent pentinyl group peak was at 2.74 (br It appears in s, 1H, -CH2CH2C≡CH), 2.37(m, 2H, -CH2CH2CH2C≡CH), 2.15(m, 2H, -CH2CH2CH2C≡CH), and 1.64(m, 2H, -CH2CH2CH2C≡CH). The ratio of EOZ:PtynOZ was determined to be 13.5:9.5. MALDI analysis showed Mn 2633Da.

[0222] Example 17. Synthesis of PEOZ 2.6K 9p(choline iodide)-3-propionate

[0223] [ka]

[0224] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.6K 9.5p (0.100 g, 0.0380 mmol, 1.0 equivalent, Mn 2633 Da) and (choline iodide)-3-azidopropionate (0.112 g, 0.342 mmol, 9 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and DMF (3 mL). After adding CuI (0.0145 g, 0.0760 mmol, 2 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50°C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.5 N aqueous HCl (1 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (9:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (0.18 g, 90% yield) as a pale yellow powder.

[0225] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6, δ) Analysis showed standard skeletal signals of PEOZ at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.32 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.94 (m, 3H, -C(=O)CH2CH3). The pendant group signals were 7.91 (br s, 1H, triazole ring, result of "click" reaction), 4.55 (m, 2H, triazole N-CH2CH2C(=O)O-), and 4.46 (m, 2H, -OCH2CH2N + (CH3)3), 3.65(m, 2H, -OCH2CH2N + (CH3)3), 3.12(s, 9H, -N +(CH3)3), and 2.62~2.58 (m, 2H, triazole N-CH2CH2C(=O)O-) were present. Furthermore, peaks of two types of pendant-type pentynyl groups appeared at 1.78 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups was, 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, the integral was determined to be 8.15, and the calculated molecular weight was 5307 Da.

[0226] Example 18. Synthesis of PEOZ 2.6K 9p NMPOH

[0227] [ka]

[0228] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.6K 9.5p (0.793 g, 0.301 mmol, 1.0 equivalent, Mn 2633Da) and (N-methyl-4-piperidyl)-3-azidopropionate (0.576 g, 2.710 mmol, 9 equivalents) were added under an argon atmosphere, and then a stirring bar and THF (8 mL) were added. After adding CuI (0.115 g, 0.602 mmol, 2 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50°C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.5 N aqueous HCl (6 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and water (9:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was dissolved in MeOH and purified by precipitation in MTBE. After filtration, the resulting precipitate was freeze-dried using a freeze-dryer to obtain the desired product (1.11 g, yield 83%) as a pale yellow powder.

[0229] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6, δ) Analysis showed standard skeletal signals of PEOZ at 7.93 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N-skeleton), 3.16 (m, 2H, -N-CH2), 2.31 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.93 (m, 3H, -C(=O)CH2CH3). The pendant group signals were 7.93 (br s, 1H, triazole ring, result of "click" reaction), 4.83 (br It was present in s, piperidine's -OCH<, 1H), 4.55 (m, 2H, triazole N-CH2CH2C(=O)O-), 2.96 (m, piperidine's CH3NCH2CH2CHO-, 4H), and 2.60 (m, 2H, triazole N-CH2CH2C(=O)O-), 2.60 (br s, CH3-piperidine, 3H), 1.95 (m, piperidine's CH3NCH2CH2CHO-, 2H), and 1.62 (m, piperidine's CH3NCH2CH2CHO-, 2H). Furthermore, peaks for two types of pendant-type pentinyl groups appear at 1.76 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.62 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 8.3, and the calculated molecular weight was 4700 Da.

[0230] Example 19. Synthesis of PEOZ 2.6K 9p NMPCA

[0231] [ka]

[0232] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.6K 9.5p (1.00 g, 0.380 mmol, 1.0 equivalent, Mn 2633Da) and (3-azidopropyl)N-methylpiperidinoate (0.774 g, 3.418 mmol, 9 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and THF (10 mL). After adding CuI (0.145 g, 0.760 mmol, 2 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 2N aqueous HCl (1.7 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and water (9:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was dissolved in MeOH and purified by precipitation in MTBE. After filtration, the resulting precipitate was freeze-dried using a freeze-dryer to obtain the desired product (1.73 g, 91% yield) as a pale yellow powder.

[0233] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6,δ) Analysis showed standard skeleton signals of PEOZ at 7.93 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.31 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.93 (m, 3H, -C(=O)CH2CH3). The pendant group signals were 7.87 (br s, 1H, triazole ring, result of "click" reaction), 4.37 (m, 2H, triazole N-CH2CH2CH2CO-), 4.01 (m, 2H, N3CH2CH2CH2O-), 2.90 (m, 2H, Pip(-CHH)2CH-C(=O)O-), and 2.65 (m, 2H Peaks for two types of pendant-type pentynyl groups appeared at 1.77 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.62 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups was as follows: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 9.0, and the calculated molecular weight was 5000 Da.

[0234] Example 20. (PEOZ) 15 Synthesis of (PtynOZ)5-OH 2.3K, (also known as PEOZ-OH 2.3K 5p)

[0235] [ka]

[0236] In a 250 mL oven-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, 16.845 g (169.6 mmol, 15 equivalents) and 7.735 g (56.49 mmol, 5 equivalents) of 2-ethyl-2-oxazoline were added under an argon atmosphere. A stirring bar and 118 mL of PhCl were then added. 1.00 mL (11.298 mmol, 1.00 equivalent) of TfOH was added dropwise, and the mixture was stirred at room temperature for 5 minutes. The reaction product was then heated to 110°C over 20 minutes and stirred at that temperature for 40 minutes. After cooling to room temperature, the polymerization mixture was transferred to an oven-dried 1000 mL round-bottom flask and concentrated using a rotary evaporator to obtain a sticky residue. After adding an aqueous solution of Na2CO3 (452 ​​mL, 0.2 M, 2.00 equivalents), residual chlorobenzene was azeotropically evaporated (approximately 100 mL in total). The resulting aqueous solution was diluted with MeCN (250 mL) and stirred overnight. After removing some of the MeCN and water using a rotary evaporator, NaCl (30.4 g, 10% w / w) was added to the resulting aqueous solution (304 g), and the product was then extracted into DCM (2 times × 300 mL). The combined organic phase was dehydrated with Na2SO4, filtered, concentrated, and further dried in vacuum at 50°C to obtain the desired product (23.2 g, yield 94.5%) as white crystals.

[0237] 1¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed a normal skeleton peak at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.32 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.96 (m, 3H, -C(=O)CH2CH3). The pendent pentinyl group peak was at 2.74 (br It appears in s, 1H, -CH2CH2C≡CH), 2.37(m, 2H, -CH2CH2CH2C≡CH), 2.15(m, 2H, -CH2CH2CH2C≡CH), and 1.64(m, 2H, -CH2CH2CH2C≡CH). The ratio of EOZ:PtynOZ was determined to be 15.73:5.25. MALDI analysis showed Mn 2279Da and PDI 1.04.

[0238] Example 21. (PrOZ) 15 Synthesis of (PtynOZ)5-OH 2.6K, (also known as PPOZ-OH 2.6K 5p)

[0239] [ka]

[0240] In a 100 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, 2-propyl-2-oxazoline (7.129 g, 63.0 mmol, 15 equivalents) and 2-pentinyl-2-oxazoline (2.881 g, 14.4 mmol, 5 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and PhCl (13 mL). TfOH (371.2 μL, 4.20 mmol, 1.00 equivalent) was added dropwise, and the mixture was stirred at room temperature for 5 minutes. The reaction was then heated to 110 °C over 20 minutes, and stirred at that temperature for 40 minutes. The polymerization mixture was then cooled to room temperature, and most of the solvent was removed using a rotary evaporator. After adding aqueous Na2CO3 solution (42 mL, 0.4 M, 4.00 equivalents), the residual chlorobenzene was azeotropically evaporated. The resulting aqueous solution was diluted with MeCN (40 mL) and stirred overnight. After removing the MeCN using a rotary evaporator, the product was extracted to DCM. The combined organic phase was dehydrated with Na2SO4, filtered, concentrated, and dried under vacuum to obtain the desired product (9.7 g, 97% yield) as white crystals.

[0241] 1 ¹H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6, δ) analysis showed a normal skeleton peak at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.27 and 2.22 (m, total area 2H, -C(=O)CH2CH2CH3), 1.48 (m, 2H, -C(=O)CH2CH2CH3), and 0.84 (m, 3H, -C(=O)CH2CH2CH3). The pendent-type pentinyl group peak was at 2.73 (br It appears in s, 1H, -CH2CH2C≡CH), 2.36(m, 2H, -CH2CH2CH2C≡CH), 2.15(m, 2H, -CH2CH2CH2C≡CH), and 1.64(m, 2H, -CH2CH2CH2C≡CH). The PrOZ:PtynOZ ratio was determined to be 16.7:5.57. MALDI analysis showed Mn 2670Da and PDI 1.05.

[0242] Example 22. Synthesis of PEOZ 2.3K 1.4p (choline iodide)-2-propionate

[0243]

Chem.

[0244] Into a 25 mL oven-dried round-bottom flask equipped with a reflux condenser and an argon gas inlet, under an argon atmosphere, PEOZ-OH 2.3K 5.2p (1.00 g, 0.439 mmol, 1.0 equivalent, Mn 2279 Da) and (choline iodide)-2-azidopropionate (0.216 g, 0.658 mmol, 1.5 equivalents) were charged, and then a stir bar and DMF (10 mL) were added. After adding CuI (0.042 g, 0.219 mmol, 0.5 equivalent), the resulting mixture was stirred at room temperature for 5 minutes and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (2.5 mL) and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (4:1). The resulting solution was concentrated using a rotary evaporator at 34 °C. The residue was purified by silica gel column chromatography using a Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the elution solvent. After removing MeOH, the resulting aqueous solution was lyophilized using a freeze dryer to obtain the desired product (1.04 g, 85% yield) as a pale yellow powder.

[0245] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6,δ) Analysis showed standard skeleton signals of PEOZ at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.75 (br s, 1H, -CH2CH2C≡CH, intact), 2.32 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.94 (m, 3H, -C(=O)CH2CH3). The pendant group signals were 7.92 (br s, 1H, triazole ring, result of "click" reaction), 5.64 (m, 2H, triazole N-CH(CH3)C(=O)O-), and 4.52 (m, 2H, -OCH2CH2N + (CH3)3), 3.66(m, 2H, -OCH2CH2N + (CH3)3), 3.05(s, 9H, -N + (CH3)3), and 1.75(d, 3H, J=7.0Hz, triazole N-CH(CH3)(C(=O)O-). Furthermore, peaks of two types of pendant-type pentinyl groups appeared at 1.79(m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64(m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups was 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 1.4, and the calculated molecular weight was 2738 Da.

[0246] Synthesis of PEOZ-OH 2.3K npcholine 3-propionate

[0247] [ka]

[0248] Example 23. Synthesis of PEOZ 2.3K 1p(choline iodide)-3-propionate (where n=1)

[0249] [ka]

[0250] Into a 25 mL oven-dried round-bottom flask equipped with a reflux condenser and an argon gas inlet, under an argon atmosphere, PEOZ-OH 2.3K 5.2p (0.80 g, 0.351 mmol, 1.0 eq, Mn 2279 Da) and (choline iodide)-3-azidopropionate (0.138 g, 0.421 mmol, 1.2 eq) were charged, and then a stir bar and DMF (8 mL) were charged. After adding CuI (0.167 g, 0.878 mmol, 2.5 eq), the resulting mixture was stirred at room temperature for 5 minutes and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (2.5 mL) and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (4:1). The resulting solution was concentrated using a rotary evaporator at 34 °C. The residue was purified by silica gel column chromatography using a Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the eluent. After removing MeOH, the resulting aqueous solution was lyophilized using a freeze dryer to obtain the desired product (0.76 g, yield 83%) as a pale yellow powder.

[0251] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6, δ) analysis showed the standard backbone signal of PEOZ at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- backbone), 3.16 (m, 2H, -N-CH2), 2.32 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.94 (m, 3H, -C(=O)CH2CH3). The signals of the pendant groups were 7.91 (br s, 1H, triazole ring, result of the "click" reaction), 4.55 (m, 2H, triazole N-CH2CH2C(=O)O-), 4.46 (m, 2H, -OCH2CH2N +(CH3)3), 3.65(m, 2H, -OCH2CH2N + (CH3)3), 3.12(s, 9H, -N + (CH3)3), and 2.62~2.58 (m, 2H, triazole N-CH2CH2C(=O)O-) were present. Furthermore, peaks of two types of pendant-type pentynyl groups appeared at 1.78 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups was, 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined that the integral was 1.0, and the calculated molecular weight was 2607 Da.

[0252] Example 24. Synthesis of PEOZ 2.3K 3p(choline iodide)-3-propionate (where n=3)

[0253] [ka]

[0254] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.3K 5.2p (0.80 g, 0.351 mmol, 1.0 equivalent, Mn 2279Da) and (choline iodide)-3-azidopropionate (0.363 g, 1.106 mmol, 3.15 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and DMF (14 mL). After adding CuI (0.167 g, 0.878 mmol, 2.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50°C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (4 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (4:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (0.93 g, yield 81%) as a pale yellow powder.

[0255] The binding of (choline iodide)-3-azidopropionate, in addition to the normal polymer backbone peaks, is observed at 7.90 (br s, 1H, triazole ring, result of "click" reaction), 4.56 (m, 2H, triazole N-CH2CH2C(=O)O-), and 4.45 (m, 2H, -OCH2CH2N + (CH3)3), 3.68(m, 2H, -OCH2CH2N + (CH3)3), 3.12(s, 9H, -N + Peaks were observed at (CH3)3) and 2.57 (m, 2H, triazole N-CH2CH2C(=O)O-). 1This was determined by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6). Furthermore, peaks for two types of pendant-type pentinyl groups appeared at 1.76 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.63 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 3.07, and the calculated molecular weight was 3263 Da.

[0256] Example 25. Synthesis of PEOZ 2.3K 5p (choline iodide)-propionate (where n=5)

[0257] [ka]

[0258] In a 50 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.3K 5.2p (0.80 g, 0.351 mmol, 1.0 equivalent, Mn 2279Da) and (choline iodide)-3-azidopropionate (0.599 g, 1.825 mmol, 5.2 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and DMF (16 mL). After adding CuI (0.167 g, 0.878 mmol, 2.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (8 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (4:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.27 g, 82% yield) as a pale yellow powder.

[0259] The binding of (choline iodide)-3-azidopropionate, in addition to the normal polymer backbone peaks, is as follows: 7.90 (br s, 1H, triazole ring, result of "click" reaction), 4.56 (m, 2H, triazole N-CH2CH2C(=O)O-), 4.46 (m, 2H, -OCH2CH2N + (CH3)3), 3.68(m, 2H, -OCH2CH2N + (CH3)3), 3.12(s, 9H, -N + Peaks were observed at (CH3)3) and 2.57 (m, 2H, triazole N-CH2CH2C(=O)O-). 1This was determined by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6). Furthermore, peaks for two types of pendant-type pentinyl groups appeared at 1.76 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.63 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, the integral was determined to be 5.03, and the calculated molecular weight was 3920 Da.

[0260] Synthesis of PEOZ-OH 2.3K np NMPCA

[0261] [ka]

[0262] Example 26. Synthesis of PEOZ 2.6K 1.5p NMPCA (where n=1.5)

[0263] [ka]

[0264] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.3K 5.2p (1.00 g, 0.439 mmol, 1.0 equivalent, Mn 2279Da) and (3-azidopropyl)-N-methylpiperidinoate (0.149 g, 0.658 mmol, 1.5 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and THF (10 mL). After adding CuI (0.0140 g, 0.073 mmol, 0.17 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.5 N aqueous HCl (5 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and 2mN HCl (2:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of 2mN HCl and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.06 g, 90% yield) as a pale yellow powder.

[0265] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6,δ) Analysis showed standard skeletal signals of PEOZ at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N-skeleton), 3.16 (m, 2H, -N-CH2), 2.32 and 2.27 (m, total area 2H, -C(=O)CH2CH3), and 0.95 (m, 3H, -C(=O)CH2CH3). The pendant group signals were 7.89 (br s, 1H, triazole ring, result of "click" reaction), 4.37 (m, 2H, triazole N-CH2CH2CH2-O-), 4.01 (m, 2H, triazole N-CH2CH2CH2-O-), 2.66 and 2.57 (m, total area 6H, piperidyl), and 2.14 (br Peaks for pendant-type pentynyl groups were present at s, 3H, pip-N-CH3), 2.03(m, 1H, pip-CH-C(O)O-), 1.99(m, 2H, -piperidyl(CHH)N-CH3), and 1.78(m, 2H, triazole N-CH2CH2CH2-O-). Furthermore, peaks for two types of pendant-type pentynyl groups appeared at 1.78(m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.63(m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups was: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 1.5, and the calculated molecular weight was 2673 Da.

[0266] Example 27. Synthesis of PEOZ 2.3K 3.3p NMPCA (where n=3.3)

[0267] [ka]

[0268] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.3K 5.2p (0.995 g, 0.436 mmol, 1.0 equivalent, Mn 2279Da) and (3-azidopropyl)-N-methylpiperidinoate (0.326 g, 1.440 mmol, 3.3 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and THF (10 mL). After adding CuI (0.0416 g, 0.218 mmol, 0.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.4 N aqueous HCl (5 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and 2mN HCl (2:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of 2mN HCl and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.31 g, 95% yield) as a pale yellow powder.

[0269] The binding of (3-azidopropyl)-N-methylpiperidinoate showed peaks at 7.89 (br s, 1H, triazole ring, result of "click" reaction), 4.37 (m, 2H, triazole N-CH2CH2CH2-O-), 4.01 (m, 2H, triazole N-CH2CH2CH2-O-), 2.58 (m, total area 6H, piperidyl), 2.13 (br s, 3H, pip-N-CH3), 2.03 (m, 1H, pip-CH-C(O)O-), 1.95 (m, 2H, -piperidyl(CHH)N-CH3), and 1.77 (m, 2H, triazole N-CH2CH2CH2-O-), in addition to the normal polymer backbone peaks. 1This was determined by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6). Furthermore, peaks for two types of pendant-type pentinyl groups appear at 1.77 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.63 (m, 2H, main chain -CH2CH2CH2C CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 3.3, and the calculated molecular weight was 3146 Da.

[0270] Example 28. Synthesis of PEOZ 2.3K 4.5p NMPCA (where n=4.5)

[0271] [ka]

[0272] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PEOZ-OH 2.3K 5.2p (0.995 g, 0.436 mmol, 1.0 equivalent, Mn 2279Da) and (3-azidopropyl)-N-methylpiperidinoate (0.447 g, 1.96 mmol, 4.5 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and THF (10 mL). After adding CuI (0.0374 g, 0.196 mmol, 0.45 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.5 N aqueous HCl (5 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and 2mN HCl (5:4). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of 2mN HCl and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.31 g, 95% yield) as a pale yellow powder.

[0273] The binding of (3-azidopropyl)-N-methylpiperidinoate showed peaks at 7.87 (br s, 1H, triazole ring, result of "click" reaction), 4.37 (m, 2H, triazole N-CH2CH2CH2-O-), 4.01 (m, 2H, triazole N-CH2CH2CH2-O-), 2.63 and 2.58 (m, total area 6H, piperidyl), 2.12 (br s, 3H, pip-N-CH3), 2.03 (m, 1H, pip-CH-C(O)O-), 1.98 (m, 2H, -piperidyl(CHH)N-CH3), and 1.78 (m, 2H, triazole N-CH2CH2CH2-O-), in addition to the normal polymer backbone peaks. 1This was determined by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6). Furthermore, peaks for two types of pendant-type pentinyl groups appeared at 1.78 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, the integral was determined to be 4.5, and the calculated molecular weight was 3461 Da.

[0274] Synthesis of PPOZ-OH 2.6K npcholine

[0275] [ka]

[0276] Example 29. Synthesis of PPOZ 2.6K 1p(choline iodide)-3-propionate (where n=1)

[0277] [ka]

[0278] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PPOZ-OH 2.6K 5.6p (1.00 g, 0.377 mmol, 1.0 equivalent, Mn 2654Da) and (choline iodide)-3-azidopropionate (0.153 g, 0.452 mmol, 1.2 equivalents, 97 wt%) were added under an argon atmosphere, and then a stirring bar and DMF (12 mL) were added. After adding CuI (0.179 g, 0.942 mmol, 2.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50°C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (3 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (4:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (0.98 g, yield 85%) as a pale yellow powder.

[0279] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6, δ) Analysis showed standard skeletal signals of PPOZ at 7.99 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.27 and 2.21 (m, total area 2H, -C(=O)CH2CH2CH3), 1.48 (m, 2H, -C(=O)CH2CH2CH3), and 0.84 (m, 3H, -C(=O)CH2CH2CH3). The pendant group signals were 7.91 (br s, 1H, triazole ring, result of "click" reaction), 4.55 (m, 2H, triazole N-CH2CH2C(=O)O-), and 4.46 (m, 2H, -OCH2CH2N + (CH3)3), 3.64(m, 2H, -OCH2CH2N+ (CH3)3), 3.10(s, 9H, -N + (CH3)3) and 2.57(m, 2H, triazole N-CH2CH2C(=O)O-) were present. Furthermore, peaks for two types of pendant-type pentynyl groups appeared at 1.78(m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64(m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups was 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined that the integral was 1.0, and the calculated molecular weight was 2982 Da.

[0280] Example 30. Synthesis of PPOZ 2.6K 3p(choline iodide)-3-propionate (where n=3)

[0281] [ka]

[0282] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PPOZ-OH 2.6K 5.6p (1.00 g, 0.377 mmol, 1.0 equivalent, Mn 2654Da) and (choline iodide)-3-azidopropionate (0.402 g, 1.187 mmol, 3.15 equivalents, 97 wt%) were added under an argon atmosphere, and then a stirring bar and DMF (10 mL) were added. After adding CuI (0.179 g, 0.942 mmol, 2.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50°C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (3 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (4:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.14 g, 83% yield) as a pale yellow powder.

[0283] The binding of (choline iodide)-3-azidopropionate, in addition to the normal polymer backbone peaks, is as follows: 7.91 (br s, 1H, triazole ring, result of "click" reaction), 4.55 (m, 2H, triazole N-CH2CH2C(=O)O-), 4.46 (m, 2H, -OCH2CH2N + (CH3)3), 3.66(m, 2H, -OCH2CH2N + (CH3)3), 3.12(s, 9H, -N + Peaks were observed at (CH3)3) and 2.57 (m, 2H, triazole N-CH2CH2C(=O)O-). 1 H NMR (Varian, 500MHz, 10mg / mL DMSO-d 6、This was determined by δ) analysis. Furthermore, peaks for two types of pendant-type pentinyl groups appear at 1.77 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is, 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 2.98, and the calculated molecular weight was 3638 Da.

[0284] Example 31. Synthesis of PPOZ 2.6K 4.7p(choline iodide)-3-propionate (where n=4.7)

[0285] [ka]

[0286] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PPOZ-OH 2.6K 5.6p (1.00 g, 0.377 mmol, 1.0 equivalent, Mn 2654Da) and (choline iodide)-3-azidopropionate (0.701 g, 2.07 mmol, 5.5 equivalents, 97 wt%) were added under an argon atmosphere, and then a stirring bar and DMF (10 mL) were added. After adding CuI (0.179 g, 0.942 mmol, 2.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50°C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (2 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and water (4:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of water and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.27 g, 80% yield) as a pale yellow powder.

[0287] The binding of (choline iodide)-3-azidopropionate, in addition to the normal polymer backbone peaks, is as follows: 7.91 (br s, 1H, triazole ring, result of "click" reaction), 4.56 (m, 2H, triazole N-CH2CH2C(=O)O-), 4.45 (m, 2H, -OCH2CH2N + (CH3)3), 3.69(m, 2H, -OCH2CH2N + (CH3)3), 3.12(s, 9H, -N + Peaks were observed at (CH3)3) and 2.57 (m, 2H, triazole N-CH2CH2C(=O)O-). 1 H NMR (Varian, 500MHz, 10mg / mL DMSO-d 6、 This was determined by δ) analysis. Furthermore, peaks for two types of pendant-type pentinyl groups appear at 1.76 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is, 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 4.7, and the calculated molecular weight was 4200 Da.

[0288] Synthesis of PPOZ-OH 2.6K np NMPCA

[0289] [ka]

[0290] Example 32. Synthesis of PPOZ 2.6K 1p NMPCA (where n=1)

[0291] [ka]

[0292] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PPOZ-OH 2.6K 5.6p (1.00 g, 0.377 mmol, 1.0 equivalent, Mn 2654Da) and (3-azidopropyl)-N-methylpiperidinoate (0.102 g, 0.452 mmol, 1.2 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and THF (10 mL). After adding CuI (0.0359 g, 0.188 mmol, 0.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.5 N aqueous HCl (3 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and 2mN HCl (3:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of 2mN HCl and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.00 g, 91% yield) as a pale yellow powder.

[0293] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6,δ) Analysis showed standard skeleton signals of PPOZ at 7.91 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.16 (m, 2H, -N-CH2), 2.27 and 2.21 (m, total area 2H, -C(=O)CH2CH2CH3), 1.48 (m, 2H, -C(=O)CH2CH2CH3), and 0.84 (m, 3H, -C(=O)CH2CH2CH3). The signal of the pendant group was 7.85 (br It was present in s, 1H, triazole ring (result of "click" reaction), 4.37 (m, 2H, triazole N-CH2CH2CH2-O-), 4.01 (m, 2H, triazole N-CH2CH2CH2-O-), 2.58 (m, total area 6H, piperidyl), 2.15 (br s, 3H, pip-N-CH3), 2.01 (m, 2H, -piperidyl(CHH)N-CH3), 1.95 (m, 1H, pip-CH-C(O)O-), and 1.64 (m, 2H, triazole N-CH2CH2CH2-O-). Furthermore, peaks for two types of pendant-type pentinyl groups appear at 1.77 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.63 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 1.09, and the calculated molecular weight was 2940 Da.

[0294] Example 33. Synthesis of PPOZ 2.6K 3p NMPCA (where n=3)

[0295] [ka]

[0296] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PPOZ-OH 2.6K 5.6p (1.00 g, 0.377 mmol, 1.0 equivalent, Mn 2654Da) and (3-azidopropyl)-N-methylpiperidinoate (0.281 g, 1.243 mmol, 3.3 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and THF (10 mL). After adding CuI (0.0359 g, 0.188 mmol, 0.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.5 N aqueous HCl (3 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and 2mN HCl (4:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of 2mN HCl and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.12 g, yield 86%) as a pale yellow powder.

[0297] The binding of (3-azidopropyl)-N-methylpiperidinoate showed peaks at 7.86 (br s, 1H, triazole ring, result of "click" reaction), 4.37 (m, 2H, triazole N-CH2CH2CH2-O-), 4.01 (m, 2H, triazole N-CH2CH2CH2-O-), 2.58 (m, total area 6H, piperidyl), 2.13 (br s, 3H, pip-N-CH3), 2.01 (m, 2H, -piperidyl(CHH)N-CH3), 1.95 (m, 1H, pip-CH-C(O)O-), and 1.77 (m, 2H, triazole N-CH2CH2CH2-O-), in addition to the normal polymer backbone peaks. 1This was determined by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6). Furthermore, peaks for two types of pendant-type pentinyl groups appeared at 1.77 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 3.32, and the calculated molecular weight was 3526 Da.

[0298] Example 34. Synthesis of PPOZ 2.6K 5p NMPCA (where n=5)

[0299] [ka]

[0300] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PPOZ-OH 2.6K 5.6p (1.00 g, 0.377 mmol, 1.0 equivalent, Mn 2654Da) and (3-azidopropyl)-N-methylpiperidinoate (0.40 g, 1.768 mmol, 4.7 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and THF (10 mL). After adding CuI (0.0359 g, 0.188 mmol, 0.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 0.5 N aqueous HCl (6 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of THF and 2mN HCl (1:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of 2mN HCl and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (1.26 g, yield 84%) as a pale yellow powder.

[0301] The binding of (3-azidopropyl)-N-methylpiperidinoate showed peaks at 7.86 (br s, 1H, triazole ring, result of "click" reaction), 4.37 (m, 2H, triazole N-CH2CH2CH2-O-), 4.01 (m, 2H, triazole N-CH2CH2CH2-O-), 2.59 (m, total area 6H, piperidyl), 2.13 (br s, 3H, pip-N-CH3), 1.98 (m, 2H, -piperidyl(CHH)N-CH3), 1.98 (m, 1H, pip-CH-C(O)O-), and 1.77 (m, 2H, triazole N-CH2CH2CH2-O-), in addition to the normal polymer backbone peaks. 1This was determined by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6). Furthermore, peaks for two types of pendant-type pentinyl groups appeared at 1.77 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups is: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 5.04, and the calculated molecular weight was 3978 Da.

[0302] Example 35. Synthesis of PPOZ 2.6K 1p spermine

[0303] [ka]

[0304] In a 25 mL furnace-dried round-bottom flask equipped with a reflux condenser and argon gas inlet, PPOZ-OH 2.6K 5.6p (0.20 g, 0.075 mmol, 1.0 equivalent, Mn 2654Da) and N1-azido-spermine-3HCl (0.0364 g, 0.106 mmol, 1.4 equivalents) were added under an argon atmosphere, followed by the addition of a stirring bar and DMF (2 mL). After adding TEA (63.1 μL, 0.452 mmol, 6 equivalents) and CuI (0.0359 g, 0.188 mmol, 0.5 equivalents), the resulting mixture was stirred at room temperature for 5 minutes, and then stirred at 50 °C for 18 hours. After cooling to room temperature, the reaction mixture was quenched by adding 1.0 N aqueous HCl (2 mL), and then stirred for 5 minutes. The mixture was passed through a Dowex® M4195 column, and the column was eluted with a mixture of DMF and 2mN HCl (1:1). The resulting solution was concentrated using a rotary evaporator at 34°C. The residue was purified by silica gel column chromatography using Biotage (SNAP Ultra C18 cartridge) and a mixture of 2mN HCl and methanol as the eluent. After removing MeOH, the resulting aqueous solution was freeze-dried using a freeze-dryer to obtain the desired product (0.15 g, 70% yield) as a pale yellow powder.

[0305] 1 H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d 6,δ) Analysis showed standard skeleton signals of PPOZ at 7.94 (br m, 1H, terminal NH), 3.34 (m, 4H, -NCH2CH2N- skeleton), 3.17 (m, 2H, -N-CH2), 2.27 and 2.21 (m, total area 2H, -C(=O)CH2CH2CH3), 1.48 (m, 2H, -C(=O)CH2CH2CH3), and 0.84 (m, 3H, -C(=O)CH2CH2CH3). The signal of the pendant group was 7.80 (br Peaks for two types of pendant-type pentynyl groups appeared at 1.78 (m, 2H, triazole ring, result of "click" reaction), 4.42 (m, 2H, triazole N-CH2CH2CH2-NH-), 2.95 (m, 2H, spermine-CH2NH2), 2.88 (m, 8H, -CH2NHCH2-), and 1.64 (m, 2H, triazole N-CH2CH2CH2-NH-). Furthermore, peaks for two types of pendant-type pentynyl groups appeared at 1.78 (m, 2H, main chain -CH2CH2CH2-triazole ring, "after click") and 1.64 (m, 2H, main chain -CH2CH2CH2C≡CH, intact). The number of pendant groups was: 1 By comparing the integrals of the pendant group protons and polymer backchain protons from 1H NMR analysis, it was determined to be 0.57, and the calculated molecular weight was 2487 Da.

[0306] Example 36: Gel shift assay of pDNA / PEOZ coramide 1K and 2K polyplexes with different polymer backbone charge numbers. Gel shift assays were performed to evaluate the binding ability of two PEOZ cationic polymers containing choline charged groups to plasmid DNA (pDNA). These were PEOZ 1K colamide (Example 7) and PEOZ 2K 5.8p colamide (Example 9). Various polymer-to-pDNA ratios (w / w) were prepared and tested on 1% agarose gels (E-Gel agarose gels, Invitrogen).

[0307] The plasmid DNA (pDNA) used was gWiz green fluorescent protein (Aldevron), encoded by the GFP gene. The plasmid had a length of 5757 bp and was supplied in water at a concentration of 5.05 μg / μL. The plasmid was diluted in 10 mM citrate buffer (pH 4) to prepare a solution at 0.1 μg / μL. Stock solutions containing PEOZ collamide polymer were prepared in ethanol (Supelco, EX0276-1) at concentrations of 31.53 mg / mL for PEOZ 1K collamide and 36.75 mg / mL for PEOZ 2K collamide. To evaluate the amount of PEOZ polymer that completely bound to the pDNA and delayed the mobility of the DNA band, 1 μg of pDNA was mixed with various μg concentrations of PEOZ collamide polymer, and the complexes were allowed to form at room temperature for 10–15 minutes. The complex mixture was diluted in ethanol to 5 ng / μL of pDNA, and 20 μL was loaded onto E-Gel EX (Invitrogen) containing 1% agarose with SYBR Gold II, and run for 10 minutes. The gel was analyzed using a gel imaging system (E-Gel Power Snap Electrophoresis System, Invitrogen) to detect shifts in the pDNA bands.

[0308] Figure 3A shows pDNA bound to PEOZ colamide 1K as polymer concentration increases from 90 to 6000 μg, and Figure 3B shows pDNA bound to PEOZ colamide 2K as polymer concentration increases from 5 to 50 μg. The gel shift compares PEOZ colamide 1K, which has one cationic charge, with PEOZ colamide 2K, which has approximately 5.8 cationic charges. As the number of cationic charges on the polymer backbone increases, the amount of PEOZ polymer required to bind a certain ratio of DNA decreases.

[0309] Example 37: Gel shift assay of pDNA / PEOZ polyplexes with the same polymer backbone charge number but different types of binding chemistry. Gel shift assays were performed to evaluate the binding ability of PEOZ 2.3K 1.4p(choline-iodide)-2-propionate (Example 22) and PEOZ-OH 2.3K 1p(choline-iodide)-3-propionate (Example 23) to form complexes with pDNA. Various polymer-to-pDNA ratios (w / w) were prepared and analyzed on agarose gels (E-Gel EX agarose gel, Invitrogen).

[0310] The plasmid DNA (pDNA) used was gWiz green fluorescent protein (Aldevron), encoded by the GFP gene. The plasmid had 5757 bp and was supplied in water at a concentration of 5.05 μg / μL. The plasmid was diluted in 10 mM citrate buffer (pH 4) to prepare a 0.1 μg / μL solution. Stock solutions containing ionizable PEOZ colamide polymer were prepared in molecular biology-grade water (Corning, 46-000-CM) at various concentrations ranging from 10 to 25 μg / μL.

[0311] To evaluate the amount of PEOZ polymer that completely binds to pDNA and delays DNA band mobility, 1 μg of pDNA was mixed with various concentrations of PEOZ polymer, and the complexes were allowed to form at room temperature for 10–15 minutes. The complex mixtures were diluted in TE buffer (pH 7.4) to 5 ng / μL of pDNA, and 20 μL was loaded onto 1% E-Gel EX (Invitrogen) containing SYBR Gold II and run for 10 minutes. The gels were analyzed using a gel imaging system (E-Gel Power Snap Electrophoresis System, Invitrogen) to detect shifts in the pDNA bands. Figure 4A shows pDNA bound to PEOZ polymer containing a 2-propionate linker as polymer concentrations increase from 10–100 μg, and Figure 4B shows pDNA bound to PEOZ polymer containing a 3-propionate linker as polymer concentrations increase from 10–100 μg. The first lane contained the pDNA control. A comparison of the two PEOZ 2.3K polymers with a single cationic charge in the diagram shows that the 3-propionate linker binds to DNA at lower concentrations than the 2-propionate linker. The 3-propionate linker has an extra carbon in the linear chain between the triazole ring and the choline charge, which is not hindered.

[0312] Example 38: Gel shift assay of pDNA polyplexes prepared with PEOZ and PPOZ N-methylpiperidinoate (NMPCA) compounds Gel shift assays were performed to evaluate the binding ability of PEOZ 2.3K 1.5p NMPCA (Example 26), PEOZ 2.3K 4.5p NMPCA (Example 28), PEOZ 2.6K 9p NMPCA (Example 19), and PPOZ 2.6K 5p NMPCA (Example 34) to pDNA. Various polymer-to-pDNA ratios (w / w) were prepared and analyzed on agarose gels (E-Gel EX agarose gel, Invitrogen).

[0313] The plasmid DNA (pDNA) used was gWiz green fluorescent protein (Aldevron), encoded by the GFP gene. The plasmid had 5757 bp and was supplied in water at a concentration of 5.05 μg / μL. The plasmid was diluted in 10 mM citrate buffer (pH 4) to prepare a 0.1 μg / μL solution. Stock solutions containing PEOZ or PPOZ polymers with NMPCA were prepared in molecular biology-grade water (Corning, 46-000-CM) at various concentrations from 10 to 25 μg / μL.

[0314] To evaluate the amount of PEOZ or PPOZ polymer that completely binds to pDNA and delays the mobility of the DNA band, 1 μg of pDNA was mixed with various concentrations of PEOZ / PPOZ NMPCA polymer, and the complexes were allowed to form at room temperature for 10–15 minutes.

[0315] The complex mixture was diluted to 5 ng / μL of pDNA in 25 mM sodium acetate buffer (pH 4.8) (Figures 5A-5B) or 10 mM citrate buffer (pH 4.0) (Figures 6A-6B). 20 μL of this mixture was loaded onto 1% E-Gel EX (Invitrogen) containing SYBR Gold II and run for 10 minutes. The gel was analyzed using a gel imaging system (E-Gel Power Snap Electrophoresis System, Invitrogen) to detect pDNA band shifts.

[0316] As the number of charges increases from 1 to 4.5 and then to 9, the amount of PEOZ polymer required to bind 1 μg of DNA decreases.

[0317] Example 39: Preparation of lipid nanoparticles containing pDNA and PEOZ-collamide polymer with different charges by hand kneading method The plasmid DNA (pDNA) used in this example was pWiz GFP plasmid DNA (Aldevron). The plasmid had 5757 bp and was supplied in water at a concentration of 5.05 μg / μL. It was diluted with 10 mM citrate buffer (pH 4.5) to prepare a solution with a concentration of 0.1 mg / mL.

[0318] Individual lipid stock solutions were prepared by dissolving PEOZ coramide 1K (Example 7), PEOZ DMAEA 1K (Example 6), and PEOZ-OH 2K 5.8p coramide (Example 9) in ethanol. Each lipid stock was mixed in ethanol with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Bachem 4005619) / cholesterol (VWR 0433) and PEOZ 2K DMA (Ser-24) stock solutions in a molar ratio of 50 / 10 / 38.5 / 1.5 to prepare each PEOZ polymer (organic lipid mix solution) in ethanol (EMD Millipore EX0276-4) at various lipid mix stock concentrations for various LNP N / P ratios (Table 1). The lipid mix stock solutions were mixed with pDNA (aqueous mixture) in a 3:1 ratio of aqueous phase to organic phase while slowly mixing with a low-speed vortex. The prepared lipid nanoparticle solution was diluted in citrate buffer (pH 4), PBS buffer (pH 7.5), or 20 mM HEPES / 5% dextrose to reduce the ethanol content in the formulation to less than 6%.

[0319] The size of the formulated PEOZ LNPs was measured using dynamic light scattering (DLS) (Zetasizer Ultra, Malvern Panalytical). The particle size and polydispersity of the PEOZ LNP formulations were typical measurements for an LNP complex (Table 1).

[0320] [Table 1]

[0321] Example 40: Preparation of lipid nanoparticles containing pDNA and PEOZ-NMP polymer by microfluidic method The plasmid DNA (pDNA) used in this example was phMGFP plasmid DNA (phMGFP-Promega E6421). The plasmid had a length of 4707 bp and was supplied at a concentration of 1 μg / μL in 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. It was diluted with 10 mM citrate buffer (pH 4.5) to prepare an aqueous solution with a concentration of 0.1 mg / mL.

[0322] Organic lipid mix stock solutions (organic solutions) of PEOZ 2.6K 9p NMPCA (Example 19) and PEOZ 2.6K 9p NMPOH (Example 18) were prepared in ethanol (EMD Millipore EX0276-4) with molar ratios of PEOZ / 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Bachem 4005619) / cholesterol (VWR 0433) and PEOZ 2K DMA lipid mixture of 50 / 10 / 38.5 / 1.5, respectively. Using a microfluidic mixing system equipped with a cartridge (Nunchuck, Unchained Labs), the pDNA aqueous solution and the lipid organic solution were mixed in a ratio of 3 parts aqueous phase to 1 part organic phase, resulting in N / P ratios of 15:1 and 14.7 for the two polymers. Various flow rates (TFR) of 15, 20, 25, and 30 mL / min were selected, and 0.25 mL fractions of the LNP mixture were collected. Each fraction was diluted in 10 mM citrate buffer to reduce the EtOH content from 25% to less than 6%. The size, polydispersity, and zeta potential of the formulated LNPs were measured using dynamic light scattering (DLS) (Zetasizer Ultra, Malvern Panalytical). The LNP particle size was less than 100 nm, and the polydispersity value was closer to 0.2, which is typical for use in microfluidic devices (Table 2).

[0323] [Table 2]

[0324] Example 41: In vitro transfection of an LNP formulation prepared from PEOZ cationic polymer and containing a DNA GFP payload using HEK293 cells. Human fetal kidney (HEK) 293 cells (ATCC) were seeded at a density of 5,000–10,000 cells / well in 200 μL of 10% FBS-containing DMEM medium (Corning) in a 96-well cell processing plate (Corning). The HEK293 plates were incubated overnight at 37°C / 5% CO2 until approximately 60–70% confluence was reached. The medium was removed from each well, the cells were washed with DPBS, and replaced with selected screening buffer. The dosage of the LNP formulation was calculated based on the pDNA GFP concentration and added to the plate wells at various doses. The plates were returned to the incubator to allow cell uptake of the formulation. After 3–4 hours, 120 μL of DMEM containing 10% FBS was added to the transfected wells, and the plates were returned to the incubator for 24–72 hours. Using an EVOS-M7000 microscope (Invitrogen), cells were analyzed at 4x and 20x magnification for cell viability and green fluorescent protein (GFP) signal expression.

[0325] LNP formulations containing the cationic polymers EOZ 2.3K 1.2p(choline-iodide)-3-propionate (Example 23), PEOZ 2.3K 1.5p NMPCA (Example 26), and PEOZ 2.6K 9p NMPCA (Example 19) were prepared using microfluidic instruments as described above. They were prepared in selected buffers and screened in vitro for GFP transfection and expression in positive cells.

[0326] The selected buffer is, 1) 10 mM citrate buffer, pH 4, CB 2) Tris-EDTA buffer, pH 7.4 3) PBS, pH 7.5 4) 20 mM HEPES-5% dextrose, pH 7.5, HBD 5% 5) 20 mM MES, pH 6.1 I used it.

[0327] Some of the LNPs in various ratios, doses, and buffers were able to transfect HEK293 cells. Positive green fluorescent cells (demonstrated by GFP in Figure 7 (N / P ratio of PEOZ 2.6K 9p NMPCA-1:7 w / w=3.9 (4x - left, 20x - right)) and Figure 8 (N / P ratio of PEOZ 2.3K 1.2p (choline-iodide)-3-propionate-1:70 w / w=8.8 (4x - left, 20x - right))) indicate cellular uptake and expression.

[0328] Example 42: Gel shift assay of saRNA / PPOZ-OH 2.6K NMPCA polyplex Gel shift assays were performed to evaluate the binding ability of ionizable PPOZ 2.6K NMPCA polymers (Examples 32-34) to form complexes with saRNA. Various ratios (w / w) of PPOZ 2.6K NMPCA to saRNA were prepared and analyzed on agarose gels (E-Gel EX agarose gel, Invitrogen).

[0329] The self-amplifying RNA (saRNA) was a highly sensitive green fluorescent protein encoded by the EGFP gene from the jellyfish Aequorea victoria (Creative Biogene, PMSAR-0001). The single-stranded RNA had a length of 8528 bp, and EGFP had a molecular weight of 27 kDa. A 1.106 μg / μL aqueous solution was diluted with 10 mM sodium acetate buffer (pH 4.5) to prepare a 0.05 μg / μL solution. A stock solution containing ionizable PPOZ 2.6K NMPCA polymer was prepared at 1.0 μg / μL in sterile filtered WFI quality water (OmniPur Millipore), and then diluted with water to various concentrations from 0 to 1.0 μg / μL.

[0330] To evaluate the amount of PPOZ polymer that completely binds to saRNA and delays its mobility, 0.05 μg / μL of saRNA was mixed with various concentrations of PPOZ polymer ranging from 0 to 0.5 μg / μL or 1.0 μg / μL. The saRNA and PPOZ polymer were mixed in a 1:1 volume ratio. The resulting mixtures were incubated at room temperature for 10 minutes, and 0.5 μg of saRNA in the polyplex was loaded onto a precast 1% agarose gel. Electrophoresis was set up and run for 10 minutes. saRNA delay was analyzed using a gel imaging system (E-Gel Power Snap electrophoresis system, Invitrogen) to detect the shift in the saRNA band.

[0331] Figures 9A–9C show saRNAs bound to PPOZ 2.6K NMPCA polymers as the polymer concentration increases. The first lane M contained naked saRNA and ran as a control. Complete binding of saRNA was observed at higher ratios of PPOZ 2.6K 1p, 3p, and 5p NMPCA to saRNA at 10:1, 3:1, and 2:1 (w / w), respectively.

[0332] Example 43: Preparation of lipid nanoparticles containing saRNA and PPOZ-OH 2.6K NMPCA The self-amplifying RNA (saRNA) was a highly sensitive green fluorescent protein encoded by the EGFP gene from the jellyfish Aequorea victoria (Creative Biogene, PMSAR-0001). The single-stranded RNA had a length of 8528 bp, and EGFP had a molecular weight of 27 kDa. The supplied 1.106 μg / μL aqueous solution was diluted with 10 mM sodium acetate buffer (pH 4.5) to prepare a solution of appropriate concentration. 1.0 mL of the aqueous saRNA solution was filled into a 10 mL sterile disposable BD syringe.

[0333] Stock solutions of PPOZ-OH 2.6K NMPCA (Examples 32-34), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Bachem 4005619), cholesterol (VWR 0433), and PEOZ 2K DMA (described in concurrently pending U.S. Patent Application No. 17 / 665,190 filed February 4, 2022, the entire disclosure of which is incorporated herein by reference) were prepared in ethanol (EMD Millipore EX0276-4). The stock solutions were mixed, resulting in the preparation of lipid mixtures at two concentrations: a high lipid concentration of 10 mg / mL and a low lipid concentration of 1.5-3.5 mg / mL, with various mol% DSPC and N / P ratios as shown in Table 3 below.

[0334] [Table 3]

[0335] A 5 mL sterile disposable BD syringe was filled with 0.3 mL of an organic solution containing an ionizable lipid mix, including PPOZ 2.6K NMPCA polymer, DSPC, cholesterol, and PEOZ 2K DMA.

[0336] As shown in Figure 10, saRNA and lipids were mixed with PPOZ 2.6K NMPCA polymer, which is capable of ionizing lipids, using a microfluidic mixing system equipped with a cartridge (Nunchuck, Unchained Labs). Both syringes were mounted on the instrument and set to prepare the mixture in a 3:1 ratio of aqueous phase to organic phase. A flow rate of 20 mL / min was incorporated into the instrument and set to collect 0.25 mL of the mixture fraction. Each fraction was diluted 10-fold with 25 mM Tris-HCl (pH 7.4) and then incubated for 1 hour to reduce the ethanol content in the formulation.

[0337] The size of formulated PPOZ LNPs was measured using dynamic light scattering (DLS) (Zetasizer Ultra, Malvern Panalytical). Figures 11A and 11B show that the average particle size of the PPOZ LNP formulation after three measurements ranged from 60 to 150 nm at low PDI. The zeta potential of PPOZ LNPs was measured by electrophoretic light scattering using Zetasizer Ultra (Malvern Panalytical). The zeta potential was measured after diluting the PPOZ LNP sample 8-fold with 25 mM Tris-HCl buffer. Figures 12A and 12B show that the recorded zeta potentials ranged from -8 to -18 mV.

[0338] Example 44: Preparation of saRNA / PPOZ-OH 2.6K NMPCA polyplex A stock solution containing ionizable PPOZ-OH 2.6K NMPCA polymer (Examples 26-28) was used in this example. A working solution was prepared at a concentration of 10.0 mg / mL in ethanol and then diluted to 6.8-8.8 mg / mL. A 1.106 μg / μL aqueous solution of EGFP saRNA was diluted with 10 mM sodium acetate (pH 4.5) to prepare a solution of 0.04-0.15 mg / mL to obtain an N / P ratio of 8.

[0339] Using a pipette, PPOZ 2.6K NMPCA in ethanol was mixed by hand with saRNA in 10 mM sodium acetate (pH 4.5) in a ratio of 3 parts aqueous phase to 1 part organic phase, and then diluted 10-fold with 25 mM Tris-HCl (pH 7.4). The resulting mixture was incubated at room temperature for 1 hour to reduce the ethanol content in the polyplex.

[0340] The size of the polyplex was measured using dynamic light scattering (DLS) (Zetasizer Ultra, Malvern Panalytical). Figure 13 shows that the average particle size of the polyplex after three measurements ranged from 200 to 500 nm.

[0341] Examples 45-48: Preparation of POZ spermine The following examples demonstrate POZ spermine prepared from various preceding examples. For example, synthesis of PEOZ 2K spermineamide from PEOZ-NHS 2K (Example 1):

[0342] [ka]

[0343] Synthesis of PEOZ 2K triazole spermine from PEOZ-propargylamide 2K (Example 4):

[0344] [ka]

[0345] P[(EOZ) m (PtynOZ) n Synthesis of PEOZ 2K(n+1) triazolespermine from ]-propargylamide 2K (Example 5):

[0346] [ka]

[0347] Synthesis of PEOZ 2K 5P (glycolic acid spermineamide) ester from PEOZ-Pip-acid 1.2K 4P (Example 3):

[0348] [ka]

[0349] Example 45. Synthesis of PEOZ 2K spermineamide

[0350] [ka]

[0351] PEOZ-NHS (200.0 mg, 0.10 mmol, 2 kDa, 1.0 equivalent) and tris-Boc-spermine (50.3 mg, 0.10 mmol, 1.0 equivalent) were added to two drum vials. 0.1 N boric acid (2 mL) was added, and the mixture was stirred to dissolve. While stirring, the pH of the solution was adjusted to 8.5 by adding 0.1 N NaOH (aqueous solution) dropwise. After stirring overnight, the resulting mixture (pH 8.42) was extracted using DCM and precipitated by adding it to diethyl ether. The resulting precipitate was filtered and dried to obtain PEOZ 2K tris-Boc-spermine.

[0352] The conjugation of Tris-Boc-spermine 1 Verification was performed by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6). In addition to polymer-related main chain peaks, an additional peak for the tris-Boc-spermine moiety was identified.

[0353] Boc-deprotection was performed by treatment with TFA (15 equivalents) in DCM. The resulting reaction mixture was concentrated using a rotary evaporator. The residue was redissolved in DCM and precipitated by addition to diethyl ether. The precipitate was filtered and dried under vacuum to obtain the desired PEOZ 2K spermine amide.

[0354] The deprotection reaction showed the disappearance of the peak corresponding to the -Boc group. 1 Verification was performed by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6).

[0355] Example 46. Synthesis of PEOZ 2K Triazole Spermine

[0356] [ka]

[0357] To a THF solution of PEOZ-propargylamide 2K (1.0 equivalent, Mn 2000 Da) and N1-azidospermine (1.0 equivalent), CuI (0.4 equivalents) and TEA (1.5 equivalents) were added. The resulting mixture was stirred at room temperature for 5 minutes, then stirred at 50°C for 18 hours to obtain a cloudy yellow solution. After cooling to room temperature, the reaction mixture was quenched by adding 0.1N aqueous HCl and stirring for 5 minutes. The mixture was passed through a Dowex® M4195 column, and THF was removed from the filtrate using a rotary evaporator. The resulting aqueous solution was stirred with dichloromethane using NaCl (5 w / v%) of the water volume. The organic phase was recovered, dehydrated with Na2SO4, filtered, and concentrated. The residue was dissolved in DCM, precipitated by adding to diethyl ether, filtered, and dried under vacuum to obtain the desired product.

[0358] The binding of N1-azido-spermine was verified by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6), which showed peaks at 5.59 ppm (m, 1H, triazole-CH2NHC(=O)-) and 8.00 ppm (s, 1H, triazole ring, result of a "click" reaction) in addition to the normal polymer backbone peak.

[0359] Example 47. Synthesis of PEOZ 2K(n+1)triazolespermine

[0360] [ka]

[0361] (EOZ) m (PtynOZ) nTo a THF solution of propargylamide (1.0 equivalent, Mn 2000 Da, where m=13 and n=7) and (N-methyl-4-piperidyl)-3-azidopropionate (n+1 equivalent), CuI (0.4 × (n+1) equivalents) and TEA (1.5 × (n+1) equivalents) were added. The resulting mixture was stirred at room temperature for 5 minutes, then stirred at 50°C for 18 hours to obtain a turbid yellow solution. After cooling to room temperature, the reaction mixture was quenched by adding 0.1N aqueous HCl and stirring for 5 minutes. The mixture was passed through a Dowex® M4195 column, and THF was removed from the filtrate using a rotary evaporator. The resulting aqueous solution was stirred with dichloromethane using NaCl (5 w / v%) of the water volume. The organic phase was recovered, dehydrated with Na2SO4, filtered, and concentrated. The residue was dissolved in DCM, precipitated by adding it to diethyl ether, filtered, and dried under vacuum to obtain the desired product.

[0362] The binding of N1-azido-spermine showed peaks at 5.59 ppm (m, 1H, triazole-CH2NHC(=O)-) and 8.00 ppm (s, 1H, triazole ring, result of a "click" reaction), in addition to the normal polymer backbone peak. 1 Verification was performed by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6).

[0363] Example 48. Synthesis of PEOZ 2K 5P (glycolic acid spermineamide) ester

[0364] [ka]

[0365] In a reaction flask, under an argon atmosphere, 1.2K (1.0 equivalent) of (EOZ)5(PrAcidOZ)4-Pip-COOH, 6.0 equivalents of glycolic acid spermineamide, and 0.1 equivalent of DMAP were added, followed by the addition of a stirring bar and DMF. After adding 6.0 equivalents of DIC, the mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered using a syringe filter, the filtrate was concentrated, and then precipitated by adding it to diethyl ether. The ether solution was decanted, and the residue was stirred with freshly added diethyl ether. The white precipitate was filtered, collected, and dried under vacuum to obtain the desired product as white crystals.

[0366] The bonding of glycolic acid spermineamide showed peaks corresponding to the CH2 and spermine moieties of glycolic acid. 1 Verification was performed by 1H NMR (Varian, 500 MHz, 10 mg / mL DMSO-d6).

[0367] The cationic POZs, LNPs and polyplexes, and pharmaceutical compositions described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, for these embodiments are intended to be illustrative of some aspects of the disclosure. Any equivalent embodiment is intended to fall within the scope of the disclosure. Indeed, in addition to those shown and described herein, various modifications of formulas and structures will be 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 referenced above are expressly incorporated herein in their entirety by reference. Any headings herein are provided solely for consistency with the structural order implied in 37 CFR § 1.77 or other methods. These headings should not limit or characterize the inventions described herein.

Claims

1. Cationic POZ, Helper lipids, Polymer lipids, and Sterol lipids Lipid nanoparticles comprising a cationic POZ of formulas I to IV: R-POZ １ - Cation (I) [In the formula, R includes an initiator, POZ １ It contains polyoxazoline polymer. 【Chemistry 1】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ For each repeating unit, m is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups; m ranges from 1 to 100; a is ran, representing a random copolymer, or block, representing a block copolymer; and T includes a terminal group. 【Chemistry 2】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ For each repeating unit, m is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, m ranges from 1 to 100, a is ran representing a random copolymer or block representing a block copolymer, and T includes a terminal group. 【Transformation 3】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ Each repeating unit is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, where m ranges from 1 to 100, a ranges from 3 to 20, and T includes a terminal group. Lipid nanoparticles selected from the group consisting of one of the following.

2. Lipid nanoparticles according to claim 1, comprising approximately 0.1% to approximately 10% cationic POZ, approximately 30% to approximately 80% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 15% to approximately 65% ​​helper lipids.

3. Lipid nanoparticles according to claim 1, comprising approximately 30% to approximately 70% cationic POZ, approximately 30% to approximately 50% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 5% to approximately 15% helper lipids.

4. The polymer lipid is a POZ-lipid of formula V. R-POZ ２ -L-lipids (V) [In the formula, R includes an initiator, POZ ２ It contains poly(oxazoline), L contains a linking group, The lipid comprises an uncharged lipid containing at least one hydrophobic moiety. ], the lipid nanoparticle according to claim 1.

5. Lipid nanoparticles according to claim 4, wherein the linking groups are physiologically degradable.

6. Lipid nanoparticles according to claim 4, wherein the linking group is stable.

7. Cationic POZ of formula I R-POZ １ - Cation (I) [wherein, R contains a starting group, and POZ １ contains a polyoxazoline polymer.], Helper lipids, Polymer lipids, and Sterol lipids Lipid nanoparticles containing lipids.

8. The polymer lipid is a POZ-lipid of formula V. R-POZ ２ -L-lipids (V) [In the formula, R includes an initiator, POZ ２ It contains poly(oxazoline), L contains a linking group, The lipid comprises an uncharged lipid containing at least one hydrophobic moiety. ], the lipid nanoparticle according to claim 7.

9. Lipid nanoparticles according to claim 8, wherein the linking groups are physiologically degradable.

10. Lipid nanoparticles according to claim 8, wherein the linking group is stable.

11. Lipid nanoparticles according to claim 7, comprising approximately 0.1% to approximately 10% cationic POZ, approximately 30% to approximately 80% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 15% to approximately 65% ​​helper lipids.

12. Lipid nanoparticles according to claim 7, comprising approximately 30% to approximately 70% cationic POZ, approximately 30% to approximately 50% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 5% to approximately 15% helper lipids.

13. Cationic POZ of formula II 【Chemistry 4】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ For each repeating unit, m is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups; m ranges from 1 to 100; a is ran, representing a random copolymer, or block, representing a block copolymer; and T includes a terminal group. Helper lipids, Polymer lipids, and Sterol lipids Lipid nanoparticles containing lipids.

14. The polymer lipid is a POZ-lipid of formula V. R-POZ ２ -L-lipids (V) [In the formula, R includes an initiator, POZ ２ It contains poly(oxazoline), L contains a linking group, The lipid comprises an uncharged lipid containing at least one hydrophobic moiety. ], the lipid nanoparticle according to claim 13.

15. Lipid nanoparticles according to claim 14, wherein the linking groups are physiologically degradable.

16. Lipid nanoparticles according to claim 14, wherein the linking group is stable.

17. Lipid nanoparticles according to claim 13, comprising approximately 0.1% to approximately 10% cationic POZ, approximately 30% to approximately 80% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 15% to approximately 65% ​​helper lipids.

18. Lipid nanoparticles according to claim 13, comprising approximately 30% to approximately 70% cationic POZ, approximately 30% to approximately 50% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 5% to approximately 15% helper lipids.

19. Cationic POZ of formula III 【Transformation 5】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ For each repeating unit, m is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups; m ranges from 1 to 100; a is ran, representing a random copolymer, or block, representing a block copolymer; and T includes a terminal group. Helper lipids, Polymer lipids, and Sterol lipids Lipid nanoparticles containing lipids.

20. The polymer lipid is a POZ-lipid of formula V. R-POZ ２ -L-lipids (V) [In the formula, R includes an initiator, POZ ２ It contains poly(oxazoline), L contains a linking group, The lipid comprises an uncharged lipid containing at least one hydrophobic moiety. ], the lipid nanoparticle according to claim 19.

21. Lipid nanoparticles according to claim 20, wherein the linking groups are physiologically degradable.

22. Lipid nanoparticles according to claim 20, wherein the linking group is stable.

23. Lipid nanoparticles according to claim 19, comprising approximately 0.1% to approximately 10% cationic POZ, approximately 30% to approximately 80% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 15% to approximately 65% ​​helper lipids.

24. Lipid nanoparticles according to claim 19, comprising approximately 30% to approximately 70% cationic POZ, approximately 30% to approximately 50% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 5% to approximately 15% helper lipids.

25. Cationic POZ of formula IV 【Transformation 6】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ Each repeating unit is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, where m ranges from 1 to 100, a ranges from 3 to 20, and T includes a terminal group. Helper lipids, Polymer lipids, and Sterol lipids Lipid nanoparticles containing lipids.

26. The polymer lipid is a POZ-lipid of formula IV. R-POZ ２ -L-lipids (IV) [In the formula, R includes an initiator, POZ ２ It contains poly(oxazoline), L contains a linking group, The lipid comprises an uncharged lipid containing at least one hydrophobic moiety. ], the lipid nanoparticle according to claim 25.

27. Lipid nanoparticles according to claim 26, wherein the linking groups are physiologically degradable.

28. Lipid nanoparticles according to claim 26, wherein the linking group is stable.

29. Lipid nanoparticles according to claim 25, wherein R comprises glycerol, pentaerythritol, polyglycerol, or a combination thereof.

30. Lipid nanoparticles according to claim 25, comprising approximately 0.1% to approximately 10% cationic POZ, approximately 30% to approximately 80% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 15% to approximately 65% ​​helper lipids.

31. Lipid nanoparticles according to claim 25, comprising approximately 30% to approximately 70% cationic POZ, approximately 30% to approximately 50% sterol lipids, approximately 0.5% to approximately 20% polymer lipids, and approximately 5% to approximately 15% helper lipids.

32. Compound of formula I R-POZ １ - Cation (I) [In the formula, R includes an initiator, POZ １ It contains polyoxazoline polymers.

33. The compound according to claim 32, wherein R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, or a substituted or unsubstituted aralkyl group.

34. Compound of formula II 【Transformation 7】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ For each repeating unit, m is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups; m ranges from 1 to 100; a is ran, representing a random copolymer, or block, representing a block copolymer; and T includes a terminal group.

35. The compound according to claim 34, wherein R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, a triazole having a bonded carboxylic acid, or a substituted or unsubstituted aralkyl group.

36. Compound of formula III 【Transformation 8】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ For each repeating unit, m is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups; m ranges from 1 to 100; a is ran, representing a random copolymer, or block, representing a block copolymer; and T includes a terminal group.

37. The compound according to claim 36, wherein R comprises hydrogen, a substituted or unsubstituted alkyl group, an alkyne-substituted alkyl group, a triazole having a bonded carboxylic acid, or a substituted or unsubstituted aralkyl group.

38. Compound of formula IV 【Chemistry 9】 [In the formula, R includes the initiator, and n ranges from 1 to 10, R ２ Each repeating unit is independently selected from unsubstituted or substituted alkyl, alkenyl, aralkyl, heterocyclylalkyl, or active functional groups, where m ranges from 1 to 100, a ranges from 3 to 20, and T includes a terminal group.

39. The compound according to claim 38, wherein R comprises glycerol, pentaerythritol, polyglycerol, or a combination thereof.

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