Polycation carrier particles

Abstract

Polycation carrier particles obtained by polymerizing one or more multiacrylate monomers with one or more hydrophilic monomers to form a crosslink. The polycation carrier particles have a net positive charge. The multiacrylate monomer and the hydrophilic monomer(s) are as defined herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 350,275, filed on June 8, 2022, the content of which is incorporated herein by reference.

[0002] This disclosure relates to the field of carrier particles for therapeutic agents, vaccines, etc., and more particularly to polycationic particles, as well as their methods and uses and their manufacture.

Background Art

[0003] In vivo delivery of proteins and genetic materials in the form of polynucleic acids such as mRNA, DNA, and siRNA has the potential for a wide range of therapeutic applications, from gene knockdown and editing to mRNA / DNA and protein - based vaccines. The success of the application of proteins and polynucleic acids for in vivo therapy depends on the ability to intracellularly deliver the desired gene cargo to target cells. This delivery of proteins and large polynucleic acids requires the use of delivery vehicles to provide stability to the cargo in circulation and often to facilitate cellular uptake by endocytosis.

[0004] Many approaches based on synthetic materials have been used to generate delivery vehicles. Most notably, lipid nanoparticle - based delivery of mRNA for the COVID - 19 vaccines developed by Pfizer and Moderna has achieved widespread clinical success. Polycations have also been investigated as synthetic materials for both nucleic acid complexation and protein delivery. Proteins are protected by polycationic carrier particles prior to endocytosis and are released from endosomes by the proton sponge effect. Advantages of synthetic polycations over lipid nanoparticles include ease of manufacture, stability, low cost, and synthetic control over the polymer structure and composition that enables the modulation of characteristics such as circulation time, release kinetics, and target cell delivery.

[0005] An important issue regarding the use of polycations in the delivery of nucleic acid materials is, for example, the associated general cytotoxicity of the polycations. Dissociation of the complex between the polycation and the encapsulated material (e.g., gene payload) after cell uptake results in the release of the encapsulated material as well as potentially cytotoxic polycations. Thus, once the particles dissociate intracellularly to release the encapsulated substance, improvements in polycation carrier particles are desired to reduce the cytotoxicity of the polycations.

Summary of the Invention

[0006] In one aspect, there are provided polycation carrier particles obtained by polymerization of an acrylate monomer for forming a crosslink and one or more hydrophilic monomers, wherein the polycation carrier particles have a net positive charge and the acrylate monomer is selected from Formula I or Formula V.

Chemical Formula

Chemical Formula

[0007] R and R’’ are each independently hydrogen, C1-C 14 linear or branched alkyl, C3-C8 cycloalkyl, or a 5- to 10-membered aryl or heteroaryl ring, and optionally terminate with a polymerizable group capable of crosslinking with one or more hydrophilic monomers, preferably an acrylate group. R’ is C2-C 14It is a linear or branched alkyl, C3-C8 cycloalkyl, or 5- to 10-membered aryl or heteroaryl ring, and R’ is terminated with at least one polymerizable group capable of crosslinking with one or more hydrophilic monomers, preferably an acrylate group. The alkyl, cycloalkyl, aryl, and heteroaryl may be optionally substituted and may be optionally interrupted by one or more oxygen, sulfur, or nitrogen atoms. Also, R2 is C2 or C3, and C3 is linear or cyclic.

[0008] In some embodiments, the polymerizable group is selected from an acrylate group, a methacrylate group, a (meth)acrylamide group, or a styrene group.

[0009] In some embodiments, the acrylate monomer of formula I is a multiacrylate monomer of formula II. [Chemical formula]

[0010] R1 is hydrogen or C1-C 14 It is a linear, branched, or cyclic alkyl, optionally substituted, optionally interrupted by one or more oxygen, sulfur, or nitrogen atoms, optionally terminated with an acrylate group, R2 is C2 or C3, C3 is linear or cyclic, and R3 is C2-C 14 It is a linear or branched alkyl, optionally substituted, and optionally interrupted by one or more oxygen, sulfur, or nitrogen atoms.

[0011] In some embodiments, the multiacrylate monomer is of formula III. [Chemical formula]

[0012] R1 and R2 are as defined above, R4 is -CH2-CH2-O- or -CH2-CH2-CH2-O-, the terminal CH2 group of R4 is bonded to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3, or 4.

[0013] In some embodiments, the multiacrylate monomer is of Formula IV.

Chemical formula

[0014] R1 and R2 are as defined above, R1’ has the same definition as R1, R2’ is C2-C3 (C3 is linear or cyclic), and m is an integer selected from 0, 1, 2, 3, or 4. In some embodiments, one or more of the nitrogen atoms in Formula IV are protonated.

[0015] In some embodiments, the acrylate monomer of Formula V is a multiacrylate monomer of Formula VI.

Chemical formula

[0016] R1 and R5 are independently hydrogen or C1-C 14 linear, branched or cyclic alkyl, optionally substituted, optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and optionally terminated with an acrylate group, R2 is C2 or C3 (C3 is linear or cyclic), R3 is C2-C 14 linear or branched alkyl, optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

[0017] In some embodiments, the multiacrylate monomer is of Formula VII.

Chemical formula

[0018] R1 and R5 are as defined above, R4 is -CH2-CH2-O- or -CH2-CH2-CH2-O-, the terminal CH2 group of R4 is bonded to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3, or 4.

[0019] In some embodiments, the multiacrylate monomer is of Formula VIII.

Chemical formula

[0020] R1 and R5 are as defined above, R2 is C2 or C3 (C3 is linear or cyclic), m is an integer selected from 0, 1, 2, 3, or 4, and R1’, R2’ and R5’ each independently share the definitions of R1, R2 and R5.

[0021] In some embodiments, the acrylate monomer is

Chemical formula

[0022] In some embodiments, the multiacrylate monomer is methyldiethanolamine diacrylate (DXL).

[0023] In some embodiments, the hydrophilic monomer is of Formula IX.

Chemical formula

[0024] R6 is -O-(CH2) p -Y, -NJK or -NJ2K + where p is an integer selected from 2, 3 or 4, and Y is OH, COOH, NJ2, NJ3 +Or defined as ONJ2, each J is independently defined as H, methyl or ethyl, K is optionally branched and optionally substituted ethyl, propyl or butyl, and R7 is hydrogen or methyl.

[0025] In some embodiments, the hydrophilic monomer is of formula X. [Chemical formula]

[0026] R8 is C2-C4 linear alkyl, X is selected from hydroxyl, carboxyl, tertiary amine, quaternary ammonium or amide, and the amine is optionally substituted with a methyl or ethyl group.

[0027] In some embodiments, one or more hydrophilic monomers are 2-hydroxyethyl acrylate (HEA) and / or N,N-(dimethylamino)ethyl acrylate (DMAEA).

[0028] In some embodiments, the polycation carrier particles have a size of 200 nm to 5 μm.

[0029] In some embodiments, the molar percent ratio of acrylate monomer to one or more hydrophilic monomers is 15 / 85 to 50 / 50.

[0030] In some embodiments, the polycation carrier particles further comprise a cargo substance complexed with the polycation carrier particles.

[0031] In one aspect, there is provided a complex comprising polycationic carrier particles complexed with cargo molecules, wherein the polycationic carrier particles are adapted to release the cargo molecules into cells, are degradable under physiological conditions, the polycationic carrier particles have charge-shifting properties, and are obtained by polymerizing one or more hydrophilic monomers with acrylate monomers of formula I or formula V as defined herein. In one embodiment, the cargo molecules are selected from nucleic acids, peptides, and proteins.

[0032] In one aspect, there is provided a method for producing polycationic carrier particles as defined herein, the method comprising providing an acrylate monomer as defined herein and one or more hydrophilic monomers as defined herein, and crosslinking the acrylate monomer with the one or more hydrophilic monomers.

[0033] In some embodiments, the crosslinking step comprises performing one of precipitation polymerization, microemulsion polymerization, dispersion polymerization, inverse suspension polymerization, or inverse emulsion polymerization.

[0034] In some embodiments, only one hydrophilic monomer is provided, and the molar percent ratio of acrylate monomer to hydrophilic monomer is 30±15 / 70±15. In one example, the one hydrophilic monomer is HEA and the acrylate monomer is DXL.

[0035] In some embodiments, two hydrophilic monomers are provided, and the molar percent ratio of acrylate monomer to the two hydrophilic monomers is 33±10 / 33±10 / 33±10. For example, the two hydrophilic monomers are HEA and DMAEA, and the acrylate monomer is DXL.

[0036] In some embodiments, the method further comprises providing a drug to be encapsulated and crosslinking the acrylate monomer with one or more hydrophilic monomers in a medium containing the drug.

[0037] In a further aspect, there is provided a method for generating a complex of a cargo substance and polycationic carrier particles, the method comprising generating the polycationic carrier particles by a method as defined herein, and complexing a negatively charged cargo substance to the positively charged polycationic carrier particles by exposing the polycationic carrier particles to a solution containing the cargo substance to obtain the complex.

[0038] In one aspect, there is provided a method for vaccine delivery to a subject in need thereof, the method comprising administering to the subject the polycationic carrier particles as defined herein, wherein the polycationic carrier particles are complexed with a vaccine agent or encapsulate a vaccine composition.

[0039] In one aspect, there is provided a method for inducing an immune response in vivo, the method comprising administering to a subject in need thereof the polycationic carrier particles as defined herein, which are complexed with an antigen or a nucleic acid encoding the antigen or encapsulate a vaccine composition.

[0040] In one aspect, there is provided a method for nucleic acid delivery for in vivo protein expression, the method comprising administering to a subject the polycationic carrier particles as defined herein, wherein the polycationic carrier particles are complexed with a nucleic acid encoding a protein.

[0041] In one aspect, there is provided a vaccine composition comprising the polycationic carrier particles as defined herein, which are complexed with an antigen or a nucleic acid encoding the antigen, and a pharmaceutically acceptable carrier or adjuvant. In one embodiment, the nucleic acid is mRNA.

[0042] Upon reading this disclosure, many further features and combinations thereof regarding improvements of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0043]

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DETAILED DESCRIPTION OF THE INVENTION

[0044] The present disclosure relates to carrier particles made from polycations that are “charge-shifting”. These charge-shifting polycations are materials that contain an initial high cationic charge density that enables complexation with materials associated with the carrier particles (e.g., vaccines, proteins, and / or nucleic acids). The carrier particles can then be subjected to cell uptake, often via hydrolysis, where a charge-shifting mechanism occurs, thereby reducing the overall cationic charge density and improving the dissociation and release of the complexed material (i.e., the cargo or payload of the polycationic carrier particle).

[0045] Traditionally, charge-shifting polycations for cell delivery have focused on the use of soluble polymers in either branched or linear polymer structures. The limitation of soluble polycations is that polyplex formation with nucleic acids is a difficult process to control the particle morphology, which can strongly affect particle uptake.

[0046] In vivo drug delivery by polymer particles requires ultimate degradation to biocompatible by-products under physiological conditions. In the case of crosslinking materials, this requires a crosslinking agent that is degradable. Typically, crosslinking agents containing disulfides, acetals, and ketals have been used as biodegradable functional groups that convert crosslinked polymer particles into soluble polymers upon degradation. Although these functional groups are biodegradable, there are limiting problems associated with them. Acetals and ketals are functional groups that can undergo acid-catalyzed hydrolysis and require a basic pH in aqueous solution to ensure the stability of the material prior to in vivo application. When carrier particles having acetal and ketal functional groups are endocytosed, the acidic pH of endosomes and lysosomes can induce the release of the contents of the particles before reaching the cytoplasm or nucleus. Thus, ketals and acetals can be less desirable functional groups for cell delivery. Furthermore, acetal and ketal crosslinking agents are typically relatively hydrophobic and thus are prone to problems regarding protein binding via water-soluble and hydrophobic interactions. Disulfides are another example of functional groups traditionally used in carrier particles. When disulfides are cleaved or degraded, they generate free thiol groups that are reactive and can potentially participate in unwanted side reactions (e.g., with proteins, DNA, etc.). Thus, disulfides can also be less desirable as functional groups in carrier particles.

[0047] The present disclosure provides polycationic carrier particles that overcome the drawbacks identified above while providing improved biocompatibility, reduced cytotoxicity, and biodegradability of the polycationic carrier particles, as well as the advantages of the resulting degradation by-products. The polycationic carrier particles of the present disclosure can decompose by hydrolysis to form acrylic acid units on the polymer, which are non-toxic and commonly used in biocompatible materials. The resulting degradation products are hydrolyzed small molecules, which are biocompatible and can be excreted out of the body over time through renal clearance. In some embodiments, the hydrolyzed small molecules are physiologically inert and preferably lack acrylic groups. The hydrolyzed small molecules can generally have a chemical structure similar to known food additives, such as dimethylethanol. To achieve these improvements, the polycationic carrier particles of the present disclosure are formed from acrylate monomers and one or more hydrophilic comonomers. The cross-linking agents of the present disclosure that form the polycationic carrier particles combine the improved performance of charge-shifting materials for polycation-based drug delivery with the biodegradability of the cross-linking agents. In one embodiment, the polycationic carrier particles can be synthesized by self-stabilized precipitation polymerization that utilizes the balance of polymer solubility and cross-linking agent loading to produce narrow-dispersion charge-shifting polycationic carrier particles.

[0048] As used herein, the term "acrylate" refers to a compound having one or more acrylate groups. In some embodiments, the acrylate monomer is a "multiacrylate" monomer having two or more acrylate groups. For example, the compound can be a diacrylate (two acrylate groups), a triacrylate (three acrylate groups), or a tetraacrylate (four acrylate groups).

[0049] As used herein, the term "polycat ion" refers to a polymer having a net positive charge and formed by the acrylate monomers and one or more hydrophilic comonomers described herein.

[0050] As used herein, the term "degradable" means that degradation occurs within a time frame of 2 hours to 2 weeks, preferably about 8 hours to 48 hours, under physiological conditions. Degradation may include hydrolysis of acrylate groups and acrylic acid groups, as well as dissociation of the polycationic polymer into small molecules.

[0051] As used herein, the term "optionally substituted" can be defined as having one or more substitutions selected from halogen (e.g., Cl, Br, F, and I), oxide, amine, amide, carboxyl, alkoxy, hydroxyl, alcohol, ester, ether, nitro, nitrile, alkyl (e.g., C1-C6), aryl (e.g., phenyl), acrylamide, and other known substituents that are biocompatible. In some embodiments, substituents can exclude phosphoric acid, sulfuric acid, and other groups that neutralize cationic charges.

[0052] In some embodiments, the acrylate monomer of the present disclosure has the formula I

Chemical formula

[0053] R is hydrogen, C1-C 14 a straight-chain or branched alkyl, C3-C8 cycloalkyl, or a 5- to 10-membered aryl or heteroaryl ring. R' is C2-C 14It is a 5- to 10-membered aryl or heteroaryl ring that can be crosslinked with a linear or branched alkyl, C3-C8 cycloalkyl, or at least one polymerizable group, preferably terminated with an acrylate group, capable of crosslinking with one or more hydrophilic monomers. The alkyl, cycloalkyl, aryl, and heteroaryl may be optionally substituted and may be optionally interrupted by one or more oxygen, sulfur, or nitrogen atoms. The polymerizable group can be selected from an acrylate group, a methacrylate group, a (meth)acrylamide group, or a styrene group. In some embodiments, R and R' can be linked to form a heterocycloalkyl containing nitrogen bonded to R and R' shown in Formula I. For example, it can form an optionally substituted C3-C8 heterocycloalkyl.

[0054] In some embodiments, R is hydrogen, C1-C 13 、C1-C 12 、C1-C 11 、C1-C 10 、C1-C9, C1-C8, C1-C7, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2 or methyl as defined. R may be linear or branched, may be optionally substituted, may be optionally interrupted by one or more oxygen, sulfur, or nitrogen atoms, and may be optionally terminated with an acrylate group. In some embodiments, R' is C2-C 14 、C2-C 13 、C2-C 12 、C2-C 11 、C2-C 10 、C2-C9, C2-C8, C2-C7, C2-C6, C2-C5, C2-C4, C2-C3 or C2, is optionally substituted, is optionally interrupted by one or more oxygen, sulfur, or nitrogen atoms, and is terminated by at least one polymerizable group.

[0055] The acrylate monomer of the present disclosure is a crosslinking agent that means having at least two polymerizable groups including an acrylate group linked to R2 in the formula shown herein. Monomers having a single polymerizable group do not allow the formation of polymer particles and do not allow the crosslinking necessary to form such particles, but instead result in linear polymers. In the preferred embodiments shown below, the acrylate monomer is a multiacrylate monomer having multiple acrylate groups that allows for improved crosslinking because having more groups that can react increases the crosslinking efficiency.

[0056] R2 is C2 or C3, i.e., -CH2-CH2-, -CH2-CH2-CH2- or cyclopropyl, preferably -CH2-CH2- or -CH2-CH2-CH2-. In some embodiments, R2 is unsubstituted. The acrylate monomer of the present disclosure has an acrylate group adjacent to an amine as shown in Formula I, and the spacer between the two groups is R2. This feature of the acrylate monomer allows for the polymerization of the acrylate monomer onto the polycation carrier particles by the reaction of the acrylate group enabled by the amine.

[0057] The amine exerts a direct inductive and nucleophilic effect when activating the ester towards hydrolysis. It also helps to attract water and hydroxide ions that react with the ester during hydrolysis and can further activate the ester carbonyl via hydrogen bonding.

[0058] In some embodiments, the acrylate monomer is Formula II

Chemical formula

[0059] R1 is hydrogen or C1-C 14It is a linear, branched or cyclic alkyl, optionally substituted, optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and optionally terminated by an acrylate group. R2 is C2 or C3, and C3 is linear or cyclic, preferably linear. R3 is C2-C 14 It is a linear or branched alkyl, which is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

[0060] In some embodiments, R1 is hydrogen, C1-C 13 C1-C 12 C1-C 11 C1-C 10 C1-C9, C1-C8, C1-C7, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2 or methyl. R1 may be linear or branched, optionally substituted, optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and optionally terminated by an acrylate group. In some embodiments, R3 is C2-C 14 C2-C 13 C2-C 12 C2-C 11 C2-C 10 C2-C9, C2-C8, C2-C7, C2-C6, C2-C5, C2-C4, C2-C3 or C2, and may be optionally substituted by one or more oxygen, sulfur or nitrogen atoms and optionally interrupted.

[0061] In some embodiments, the multiacrylate monomer is of formula III.

Chemical formula

[0062] R1 and R2 are as defined above. R4 is -CH2-CH2-O- or -CH2-CH2-CH2-O-, and the terminal CH2 group of R4 is bonded to the oxide of the terminal acrylate group. n is an integer selected from 1, 2, 3, or 4.

[0063] In some embodiments, the multiacrylate monomer is of Formula IV.

Chemical formula

[0064] R1 and R2 are as defined above. R1’ independently follows the same definition provided herein for R1. R2’ is C2-C3, and m is an integer selected from 0, 1, 2, 3, or 4. R2’ can be -CH2-CH2-, -CH2-CH2-CH2- or cyclopropyl, preferably -CH2-CH2- or -CH2-CH2-CH2-. In some embodiments, R2’ is unsubstituted.

[0065] In some embodiments, the acrylate monomer is of Formula V.

Chemical formula

[0066] R, R’, and R2 are as defined above. R’’ independently follows the same definition as R. In some embodiments, two or more of R, R’, and R’’ can be linked to form a nitrogen-containing heterocycloalkyl bonded to R, R’, and R’’ shown in Formula V. For example, an optionally substituted C3-C8 heterocycloalkyl can be formed.

[0067] In some embodiments, the acrylate monomer of Formula V can be a multiacrylate monomer. In some embodiments, the multiacrylate monomer is of Formula VI. [Chemical formula] is as follows.

[0068] R1, R2, and R3 are as previously defined. R5 is independently in accordance with the definition of R1 provided above.

[0069] In some embodiments, the multiacrylate monomer is Formula VII [Chemical formula] as follows.

[0070] In the formula, R1, R4, R5, and n are as previously defined.

[0071] In some embodiments, the multiacrylate monomer of Formula IV can be protonated and have one or both of the nitrogen atoms bonded to the groups R5 and R5’, both of which are independently in accordance with the definition of R1 provided above. For example, the multiacrylate monomer of Formula IV may have both nitrogens protonated, as shown in Formula VIII below. [Chemical formula] is as follows.

[0072] R1, R1’, R2, R2’, R5, R5’, and m are as previously defined.

[0073] In a preferred embodiment, the acrylate monomer is [Chemical formula] selected from.

[0074] In one embodiment, the acrylate monomer is N-methyldiethanolamine diacrylate, called (DXL) having the following formula. [Chemical formula]

[0075] The acrylate monomer can be a cationic charge shift monomer that contributes to the net positive charge and charge shift characteristics of the resulting polycation. The acrylate monomer is crosslinked or polymerized with one or more hydrophilic monomers to obtain polycation carrier particles. The acrylate monomer is crosslinked with one or more hydrophilic monomers to reduce the crosslink density of the resulting polycation. There are many techniques for polymerizing this monomer, including precipitation polymerization, microemulsion polymerization, dispersion polymerization, inverse suspension polymerization, and inverse emulsion polymerization (i.e., water-in-oil).

[0076] In some embodiments, the hydrophilic monomer is a neutral polar monomer. In some embodiments, the hydrophilic monomer has a molecular weight of less than 500 g / mol, less than 400 g / mol, or less than 300 g / mol. Smaller molecules are advantageous in that the hydrophilic monomer is more soluble in the aqueous phase. In one example, the hydrophilic monomer can be a monoacrylate monomer, a monomethacrylate monomer, an acrylamide monomer (e.g., a monoacrylamide monomer), or a methacrylamide monomer (e.g., a monomethacrylamide monomer). In some embodiments, the hydrophilic monomer can have a net positive cation charge and thus contribute to the overall charge of the polycation carrier particles. By using different hydrophilic monomers and different concentrations of hydrophilic monomers, the overall net charge of the polycation carrier particles can be adjusted, which in turn plays a role in the release kinetics and binding properties of the polycation carrier particles.

[0077] In some embodiments, the hydrophilic monomer is of formula IX. [Chemical formula] is as follows.

[0078] R6 is -O-(CH2) p -Y, -NJK or -NJ2K + where p is an integer selected from 2, 3 or 4. Y is defined as OH, COOH, NJ2, NJ3 + or ONJ2, each J is independently defined as H, methyl or ethyl, and K is ethyl, propyl or butyl (optionally branched and optionally substituted). R7 is hydrogen or methyl.

[0079] In some embodiments, the hydrophilic monomer is a monoacrylate monomer of formula X.

Chemical formula

[0080] R8 is C2-C4 linear alkyl, and X is selected from hydroxyl, carboxyl, tertiary amine, quaternary ammonium or amide. The amine may be substituted with a methyl group or an ethyl group.

[0081] In some embodiments, the acrylate monomer is crosslinked with one or more hydrophilic monomers in a molar percent ratio of 15 / 85 to 50 / 50, 20 / 80 to 40 / 60, or 25 / 75 to 35 / 65. In some embodiments, only one type of hydrophilic monomer is used, and the molar percent ratio of acrylate monomer to hydrophilic monomer is about 30 / 70. In one embodiment, two types of hydrophilic monomers are used, and the acrylate monomer to hydrophilic monomer ratio is about 33 / 33 / 33. As used herein, the term "about" can be defined as ±15, ±10, ±9, ±8, ±7, ±6, ±5, ±4, ±3, ±2 or ±1, and "±" is applied to each value or the total value of the ratios defined by the ratio.

[0082] In some embodiments, the synthesis of the charge-shifting polycation carrier particles is performed using the diacrylate crosslinker DXL. The synthesis can be a precipitation polymerization to produce degradable polycation carrier particles. DXL is an example of a diacrylate crosslinker that can be used to form degradable polymer particles due to its hydrolytically labile ester groups. Similar acrylate crosslinkers incorporating labile esters with adjacent amino groups (e.g., according to Formulas I - VIII) can also be used to prepare degradable particles and polymeric materials. As shown in the above Formulas I - VIII, examples of DXL derivatives include various spacer lengths including tertiary or secondary amino groups, and triacrylate analogs, tetraacrylate analogs. The loading percentage of DXL can be used to form particles having various sizes and various degradation rates. Copolymerizing DXL with analogs of DXL can also be used to adjust the overall rate of particle degradation.

[0083] The formation of polymer particles of controlled size can enable the formation of polyplex particles (cationic particles + payload) of controlled size. Heterogeneous polymerization systems for obtaining polymer particles include emulsion polymerization, dispersion polymerization, suspension polymerization, and precipitation polymerization. Typically, these heterogeneous polymerizations require the use of surfactants or steric stabilizers for particle formation. These surface stabilizers can leave undesirable chemical patterns on the particle surface groups that can interfere with potential delivery applications.

[0084] Precipitation polymerization is an exemplary method for obtaining polycation carrier particles on a laboratory scale, although other methods are also contemplated for industrial scale and the like. Precipitation polymerization is a high-yield method that can produce monodisperse, swellable, stabilizer-free particles suitable for use in biomedical applications. Precipitation polymerization is well-suited for producing particles containing reactive monomer(s), particularly those sensitive to water, and for producing narrowly dispersed micron-sized particles that do not contain stabilizers or surfactants. In one embodiment, the yield is defined as the weight or molar ratio of the starting monomer and optionally an initiator to the monomer present in the polymer being formed. In another embodiment, the yield is defined as the weight or molar ratio of the starting monomer and optionally an initiator to the monomer present in the particles. In various embodiments, the yield of precipitation polymerization can be at least 1%, at least 2%, at least 3%, at least 5%, preferably at least 10% or at least 20%. The polycation carrier particles can be obtained by precipitation polymerizing an acrylate monomer with one or more hydrophilic monomers under particle-forming conditions, followed by optional hydrolysis and / or functionalization. The pendant acrylate groups of the acrylate monomer and / or hydrophilic monomer can be functionalized with small molecule thiols or amino compounds.

[0085] Precipitation polymerization is initiated with a homogeneous solution of monomer (acrylate monomer and one or more hydrophilic monomers) and an initiator. A typical total monomer loading is about 1 to about 20 wt% of the solution, or about 2 to about 10 wt%, and the solvent has suitable solubility characteristics for the polymer being formed. Particle formation becomes inefficient and is limited by a total monomer loading of less than 1 wt%. In one embodiment, one or more additional monomers (i.e., monomers that are neither acrylate monomers nor hydrophilic monomers as defined herein) may be added to the monomer loading to produce a polymer tailored for a particular application.

[0086] The solvent for precipitation polymerization should be a sufficiently poor solvent to aggregate the polymer to form particles, but should be a sufficiently good solvent to swell the polymer chains on the particle surface to prevent particle-particle aggregation during polymerization. In one embodiment, the solvent used has a Hildebrand solubility parameter that is about 4 to about 5 MPa 1 / 2 higher or lower (i.e., more polar or less polar) than the Hildebrand solubility parameter of the forming polymer. Further, in some embodiments, the viscosity of the solvent is a further factor to be considered in the selection of the solvent. A low viscosity solvent is preferred. In one embodiment, the solvent has a viscosity of less than about 0.5 cP at 20 °C. The solvent used for precipitation polymerization should have a boiling point higher than the polymerization temperature (typically 60 - 70 °C in thermal initiation polymerization) and should not substantially react with the monomer or initiator. In the case of acrylate monomers, nucleophilic solvents such as water, alcohol or amine should be avoided. The particles can also be obtained from photoinitiated precipitation polymerization, which allows the use of solvents with lower boiling points. Examples of solvents suitable for the precipitation polymerization of the present disclosure include, but are not limited to, heptane, toluene, xylene, methyl ethyl ketone (MEK), tetrahydrofuran (THF), acetonitrile, ethyl acetate, benzene, cyclohexane, chloroform, or mixtures thereof. In the case of photoinitiated polymerization, solvents such as acetone, diethyl ether, dichloromethane and pentane can be used.

[0087] Alternative methods for obtaining polycation carrier particles by precipitation polymerization include inverse emulsion polymerization, microemulsion polymerization, inverse suspension polymerization, and dispersion polymerization. Suspension and inverse suspension polymer particles are formed essentially by minibulk polymerization and thus have homogeneous particle characteristics. Suspension and inverse suspension polymerization, which are carried out using mechanical dispersion of a liquid particle-forming phase (e.g., a monomer mixture) in a bulk continuous phase, usually produce particles with a broad size distribution, considering the statistical balance of droplet sharing and coalescence found in these processes. Inverse suspension polymerization of droplets of an aqueous solution of a hydrophilic monomer mechanically dispersed in a immiscible oily medium can form spherical microparticles and microgels on a large scale, although with a broad size distribution. Similarly, dispersing an aqueous coacervate phase in a continuous aqueous phase and subsequently crosslinking the dispersed droplets to form hydrogel beads can be seen as an example of aqueous-aqueous suspension polymerization that results in spherical crosslinked hydrogel particles with a large particle size distribution. Emulsion-type polymerization uses particle initiation in a continuous medium and can result in the large-scale production of narrow-dispersion nanoparticles. In both suspension and emulsion-type polymerization, water is typically used as a solvent, which cannot accept reactive and hydrolysis-labile monomers. Dispersion polymerization is initiated with a solution of a monomer, an initiator, and a colloidal stabilizer in a polymer-poor solvent. This process can be used to form monodisperse microparticles when it takes advantage of the decreasing solubility of growing polymer chains and a large amount of steric stabilizer is used to prevent aggregation of the forming particles.

[0088] The obtained polycation carrier particles can have a size of 200 nm to 5 μm. This size range is particularly suitable for the internalization of polycation carrier particles in cells (e.g., mammalian cells). The positive charge of the polycation carrier particles also plays a role in promoting cell uptake. The preferred range of the particle size is 0.5 μm to 5 μm, 1 μm to 5 μm, or 1 μm to 3 μm. The size can be defined as the diameter when the particle is an ellipsoid of revolution, or when the shape is irregular, it can be defined as the maximum distance between two opposing points taken on a line passing through the center of the particle. The polycation carrier particles of the present disclosure generally have a spherical shape with a smooth surface or a rough surface. In one embodiment, the shape is a sphere or an irregular sphere. An irregular sphere can be defined as having small protrusions on the surface, thereby making the surface rough.

[0089] When the polycation carrier particles are internalized by cells, the polycation carrier particles are exposed to specific aqueous or pH conditions that decompose the polycation carrier particles, for example, through a chemical reaction of the reactive ester of the polycation carrier particles. Degradation by hydrolysis of the reactive ester of the acrylate monomer can easily occur at pH 7, but can also occur at other pH values. Generally, faster degradation occurs at higher pH (e.g., above 7), and slower degradation occurs at lower pH (e.g., below 7). The composition of the polycation carrier particles can be varied by copolymerizing the above different acrylates and hydrophilic monomers. Hydrophilic monomers can be used to change the physical and chemical properties of the particles. Hydrophilicity, hydrophobicity, charge, degradation rate.

[0090] When the polycation carrier particles decompose, the crosslinks that held the polymer chains together in the particles are broken, and the polymer chains are released into the aqueous solution. The polymer chains consist of the acrylate monomers and some of the small molecules that were decomposed here, as described above. The polymer chains generated by particle decomposition can be processed and removed from the subject's body via renal clearance. Degradation by hydrolysis can occur under physiological conditions, i.e., at 37 °C and pH 7, without the need for further harsh external stimuli. Degradation does not occur by free radicals. Therefore, the degradation is considered to be biocompatible. When DXL is an acrylate monomer, the resulting polymer by-products are acrylic acid copolymers, which are materials often used in biomaterial applications (i.e., dental implants, hydrogels).

[0091] The substance complexed with the polycation carrier particles can be a vaccine, a genetic material (e.g., a poly-nucleic acid (mRNA, DNA, siRNA)), or a therapeutic agent. The polycation carrier particles can be used to deliver a cargo substance in an aqueous environment (e.g., intracellular) where degradation can occur. The polycation carrier particles can be used to bind proteins such as ovalbumin and other proteins such as growth factors. Negatively charged cargo substances can readily associate with the positively charged particles, preferably (e.g., nucleic acids). The size of the particles, the charge of the particles, and the composition of the particles (monomer selection) can be varied to be optimized for a particular delivery route (e.g., intravenous or oral), as well as for the delivery and dissociation of the particles in a particular environment (e.g., pH range).

[0092] In some embodiments, the polycation carrier particles can associate or encapsulate a vaccine composition and thus act as a delivery platform for an antigen. Thus, the polycation carrier particles are ● able to act as a carrier for an antigen, e.g., an active harmless virus such as an adenovirus modified to express an RNA, DNA, protein, viral capsid fragment, whole inactivated viral capsid, or desired antigenic protein containing m-RNA. ● It has a cationic group that can electrostatically bind to the antigen during storage and administration to the recipient's immune system. ● It has adjuvant properties that ensure recognition and processing by the host immune system, including cases where the adjuvant properties are based on a cationic group or polycationic group, or a specific carbohydrate group. ● It has a composition in which a cationic group or polycationic group can be cleaved by spontaneous hydrolysis or enzyme-mediated hydrolysis to release the bound antigen payload over a beneficial time frame for eliciting a strong immune response in the recipient. ● It has a cross-linking agent that can undergo slow spontaneous or enzyme-mediated hydrolysis to ensure the final clearance of the microparticles from the recipient through a process including renal clearance. ● It has a composition containing cationic groups, polycationic groups, and non-stoichiometric polyelectrolytes that can bind to the antigen for storage at room temperature defined up to 40°C and does not require cold chain logistics during storage and transportation. ● Silver nanoparticles may be further loaded to enhance the cellular immune response, for example, by co-precipitation during precipitation polymerization, or by reduction precipitation from silver salts as part of post-functionalization, or by adsorption of pre-formed silver nanoparticles onto the described polymer nanoparticles.

[0093] Thus, in some embodiments, there is provided a vaccine composition or therapeutic composition comprising the polycationic carrier particles of the present disclosure. The polycationic carrier particles of the present disclosure can be administered to a subject in need thereof without a further carrier. For example, the polycationic carrier particles can be suspended in a solution for intravenous injection. Other injection routes, particularly for vaccine applications, include intramuscular, intranasal, and transdermal (microarray needle) administration. In other embodiments, the polycationic carrier particles may be formulated as part of an oral composition (e.g., an oral capsule).

[0094] In some embodiments, the polycation carrier particles are complexed with a cargo or payload that induces an immune response and administered to a subject. The cargo of the polycation carrier particles can be a nucleic acid such as DNA and RNA. Thus, in other embodiments, a method of delivering a nucleic acid to a cell is provided by administering the polycation carrier particles of the present disclosure having a nucleic acid payload. Thus, the polycation carrier particles can be used in gene editing applications such as gene knockdown and gene editing by clustered regularly interspaced short palindromic repeats (CRISPR). In some embodiments, a method of performing gene therapy on a subject in need thereof is provided, including administering to the subject the polycation carrier particles of the present disclosure having a nucleic acid payload, for example in plasmid form.

[0095] The polycation carrier particles of the present disclosure can also be used in antibacterial materials such as antibacterial surfaces and coatings, and also in wastewater management as a flocculant.

Examples

[0096] Materials N-Methyldiethanolamine (≥99%), triethylamine (≥99%), acryloyl chloride (≥97%), 2-hydroxyethyl acrylate (HEA, 96%), N,N-(dimethylamino)ethyl acrylate (DMAEA, 98%), methyl ethyl ketone (MEK, ≥99.0%), heptane (99%), 1,4-dioxane (≥99%), acetone (≥99.5%), sterile filtered water, heavy water (D2O, 99.9%D), and deuterated chloroform (CDCl3, 99.9%D) were purchased from Sigma Aldrich. Azobisisobutyronitrile (AIBN, 99.8%) was purchased from DuPont. Dulbecco's Modified Eagle Medium (DMEM) (high glucose, sodium pyruvate), fetal bovine serum (FBS) (heat inactivated, from Canada), NucBlue Live Cell Stain ReadyProbes reagent (Hoechst33342), penicillin streptomycin (10,000 U / mL), ovalbumin fluorescein conjugate (OVA-FITC), ovalbumin Texas Red™ conjugate (OVA-RED), 1× phosphate buffered saline (PBS), and paraformaldehyde (16% (w / v) methanol-free aqueous solution) were purchased from Thermo Fisher Scientific. CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay was purchased from Promega. 96-well glass bottom plates with high performance #1.5 cover glasses were purchased from Cellvis. Hydrochloric acid (HCl) aqueous solution (35 - 37 wt%), sodium hydroxide (reagent grade), and disodium hydrogen phosphate (≥98%) were purchased from Caledon Laboratory Chemicals. Sodium chloride (≥99%) and sodium bicarbonate (≥99.7%) were purchased from ACP Chemicals. Sodium dihydrogen orthophosphate (guaranteed grade) was purchased from BDH Chemicals. Ethyl alcohol (95% v / v) was purchased from commercially available alcohol.

[0097] Synthesis of methyldiethanolamine diacrylate (DXL). N-Methyldiethanolamine (6 g, 50.4 mmol) and triethylamine (TEA) (12.714 g, 125.9 mmol) were dissolved in 400 mL of dichloromethane (DCM) in a round-bottom flask equipped with a magnetic stir bar and maintained at 0 °C in an external ice-water bath. Acryloyl chloride (11.393 g, 125.9 mmol) was added dropwise to the stirred reaction mixture over 5 - 10 minutes, whereupon the reaction mixture became opaque and changed to yellow upon addition. After the addition was complete, the reaction was stirred overnight and warmed to room temperature. The reaction mixture was washed four times with an equal volume fraction of 5 wt% sodium bicarbonate and subsequently with brine (saturated sodium chloride solution). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to give an orange oil in 90 - 95% yield. 1 1H nuclear magnetic resonance (NMR) was performed and the spectrum is shown in Figure 1. (CDCl3, 600 MHz) δ 6.37 (2H, dd), 6.10 (2H, dd), 5.80 (2H, dd), 4.23 (4H, t), 2.74 (4H, t), 2.35 (3H, s).

[0098] Hydrolysis of DXL A suspension of DXL was prepared at 0.75 wt% in 100 mM phosphate-buffered D2O, adjusted to pH 7.30, and then the monomer was dissolved in the buffer. The solution was transferred to a 5 mm NMR tube, 1 1H NMR spectra were recorded at various time intervals using a 500 MHz spectrometer from Bruker while maintaining the sample at room temperature ~22 °C. The hydrolysis of DXL was measured by calculating the ratio of the alcohol by-product to the diacrylate ester by comparing the integrals of the N-methyl (-CH3) signals of DXL (δ 2.64 ppm), 2-((2-hydroxyethyl)(methyl)amino)ethyl acrylate (δ 2.93 ppm), and N-methyldiethanolamine (δ 2.97 ppm) in the spectra recorded at different times (Figure 2). The following Scheme 1 shows the small molecule hydrolysis reaction scheme of DXL to form the corresponding alcohol by-product and acrylic acid. Scheme 1.2 - Ester hydrolysis of DXL to form ((2 - hydroxyethyl)(methyl)amino)ethyl acrylate, N - methyldiethanolamine, and acrylic acid as a by - product [Chemical formula]

[0099] Synthesis of DXL cross - linked particles by precipitation polymerization HEA - DXL (HD) particles: A mixture containing 25 - 30 mol% of DXL was obtained by combining 2 - hydroxyethyl acrylate (HEA) and methyldiethanolamine diacrylate (DXL). This mixture was dissolved in a solvent mixture of methyl ethyl ketone (MEK) and heptane with a relative volume ratio of 70 - 85% MEK at a 5% monomer loading, together with 1 mol% of AIBN as a free - radical initiator for either thermal or photolytic decomposition. A sample polymerization mixture with a total monomer loading of 1 g, targeting 70 mol% of HEA and 30 mol% of DXL in a 75 / 25 MEK / heptane solvent mixture, was provided as follows. HEA (0.5438 g, 4.684 mmol), DXL (0.4562 g, 2.007 mmol), AIBN (0.0110 g, 0.0669 mmol), MEK (11.4713 g, 14.25 mL), heptane (3.2490 g, 4.75 mL) were mixed in a 20 mL glass scintillation vial, placed in an HB - 1000 hybridizer oven (UVP) set at 70 °C, rotated and mixed at a speed of 7 rpm, and the total heating time was 5 - 8 hours. Alternatively, the reaction vessel was placed on a set of steel rollers (VIVO Electric 12 - hot dog and 5 - roller grill cooker; hotdg - v005), set at 3.25 rpm, and irradiated with two F15T8 / BL fluorescent tube light sources (15 W) at a distance of 1.8 cm from the vial at an ambient temperature of 22 - 25 °C for a total of 20 - 24 hours.

[0100] After coincidence, the reaction mixture was transferred to a 50 mL centrifuge tube and diluted with an equal volume of 1,4-dioxane. Then, the suspension was centrifuged at 130 rcf for 15 minutes, and subsequently the supernatant was removed and the particle pellet was resuspended in 1,4-dioxane. This washing procedure was repeated a total of 3 times to remove soluble polymers, residual monomers, and initiators. For samples prepared by thermal polymerization, the particles were sonicated for 20 - 40 minutes to separate the particles from larger aggregates. The dried particles were isolated as an orange solid, and the isolation yield was 5 - 10%.

[0101] HEA-DMAEA-DXL (HDD) particles: N,N-(dimethylamino)ethyl acrylate (DMAEA), DXL, and HEA were mixed with 1 mol% AIBN as a free radical initiator for either thermal decomposition or photolysis at a relative monomer feed molar ratio of 1:1:1 D:D:H. Then, they were dissolved at a monomer loading of 5% in a solvent mixture of methyl ethyl ketone (MEK) and heptane with a relative volume ratio of 70 - 95% MEK. A sample polymerization mixture targeting a molar ratio of H1:1:1 D:H in a 75 / 25 MEK / heptane solvent mixture with a total monomer loading of 1 g was provided as follows. DMAEA (0.2943 g, 2.055 mmol), DXL (0.4671 g, 2.055 mmol), HEA (0.2386 g, 2.055 mmol), AIBN (0.0101 g, 0.0609 mmol), MEK (11.4713 g, 14.25 mL), heptane (3.2490 g, 4.75 mL) were polymerized and isolated as described above for HD particles. The dried HDD particles were obtained as an orange solid with an isolation yield of 1 - 5%.

[0102] Optical microscopy protocol Using a Nikon Eclipse LV100ND upright microscope, a Nikon TiEclipse inverted microscope, and a Nikon A1 confocal Ti Eclipse microscope, HEA-DXL (HD) particles were imaged by bright-field microscopy. Images were analyzed using Nikon NIS-elements Advanced Research software version 5.11.01. Size measurements (n = 70) were manually determined using the three-point circle method.

[0103] Particle degradation protocol DXL crosslinked particles were prepared at 0.1 wt% in PBS at pH 7.4 and 0.1 M NaOH (pH ~11) to monitor particle degradation under physiological and accelerated conditions, respectively. The suspensions were maintained at room temperature and imaged by bright-field microscopy at various time points.

[0104] Loading of particles with OVA (ovalbumin) (OVA-FITC (fluorescein isocyanate), OVA-RED) The HD particles were sterilized by resuspending them at approximately 0.2 wt% in 70% ethanol for 10 minutes. After sterilization, a particle solution was prepared and handled using aseptic techniques within a guaranteed Class A2 biosafety cabinet. The particle suspension in ethanol was then centrifuged at 130 rcf for 15 minutes, followed by removal of the supernatant and subsequent resuspension in sterile water. The washing procedure was repeated a total of 2 times to remove residual ethanol and form a sterile suspension of particles in PBS at a final concentration of 0.2 wt%. Separately, a solution of 0.2 wt% OVA-FITC was prepared in PBS and sterile filtered using a 0.22 mm syringe filter. To load OVA-FITC onto the HD particles, 400 μL of 0.2 wt% HD particles in PBS was transferred to a microcentrifuge tube containing 400 μL of 0.2 wt% OVA-FITC in PBS. The suspension was mixed by pipetting 10 times, followed by vortex mixing for 1 minute. The suspension was then centrifuged at 3200 rcf for 1 minute, the supernatant was removed, and the pellet was resuspended in 800 μL of PBS. The washing procedure was repeated a total of 2 times, and the pellet was resuspended in 800 μL of PBS to form HD particles loaded with 0.1 wt% target concentration of OVA-FITC (HD-OVA-FITC). The HD-OVA-FITC particles were then imaged by bright-field and fluorescence confocal microscopy to visualize the binding of OVA-FITC onto the HD particles. The HD particles were loaded with OVA-RED in the same manner as outlined previously for OVA-FITC.

[0105] Cell Uptake Protocol RAW 264.7 macrophage cells (ATCC) were cultured in a T-75 tissue culture-treated flask in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum and 1% v / v penicillin-streptomycin (100 units / mL) and maintained at 37 °C in a humidified atmosphere containing 5% CO2. For the cell uptake assay, RAW 264.7 cells were seeded at a density of 8000 cells / well in a glass-bottom 96-well plate (Cellvis) with 100 μL of volume of supplemented medium. The cells were cultured for 3 days to reach approximately 80% confluence. The medium was removed from the wells and the cells were washed once with approximately 250 μL of PBS. To the washed cells, 100 μL of a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin was added. As controls, the cells were exposed to a) 100 μL of serum-free DMEM, b) 10% v / v DMSO in 100 μL of serum-free DMEM, and c) 0.1 wt% OVA-FITC in 100 μL of serum-free DMEM and added to the cells. The cells were incubated at 37 °C, 5% CO2 in a humidified atmosphere for 4 hours. After incubation, the supernatant was removed from the cells, washed twice with approximately 250 mL of PBS, and subsequently 4% v / v paraformaldehyde in 100 μL of sterile water was added. After 15 minutes, the paraformaldehyde solution was removed, the cells were washed twice with approximately 250 μL of PBS, and then 100 μL of PBS was added to the fixed cells. The cells were then imaged by bright-field and confocal fluorescence microscopy.

[0106] MTS assay protocol (i.e., [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]) RAW 264.7 macrophages (ATCC) were cultured in T-75 tissue culture-treated flasks in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum and 1% v / v penicillin-streptomycin (100 units / mL) and maintained at 37 °C in a humidified atmosphere containing 5% CO2. For the cell uptake assay, RAW 264.7 cells were seeded at a density of 8000 cells / well in a volume of 100 μL of supplemented medium in three tissue culture (TC)-treated 96-well plates. The cells were cultured for 3 days to reach approximately 80% confluence. The medium was removed from the wells and the cells were washed once with approximately 250 μL of PBS. To the washed cells, 100 μL of a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin was added. As controls, 100 μL of serum-free DMEM supplemented with 1% penicillin-streptomycin, 100 μL of 10% v / v DMSO in serum-free DMEM supplemented with 1% penicillin-streptomycin, 100 μL of 0.1 wt% OVA-FITC in serum-free DMEM supplemented with 1% penicillin-streptomycin, and 100 μL of a 0.1 wt% HD particle suspension in serum-free DMEM supplemented with 1% penicillin-streptomycin were added to the cells. The cells were incubated at 37 °C, 5% CO2 in a humidified atmosphere for 4 hours. 20 μL of MTS solution (Promega) was added to the wells and mixed. The plates were shaken for 10 seconds at a horizontal distance of 1 mm and read at 490 nm using a Biotek™ 800 TS absorbance reader.

[0107] Statistical analysis Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) with Bonferroni post hoc analysis. The significance level was set at p < 0.05.

[0108] Hydrolysis of DXL To model the hydrolysis and degradation of DXL crosslinked particles, the hydrolysis of DXL alone was carried out. The degradation of DXL can occur at two reactive ester sites per DXL unit, and it should be noted that the hydrolysis of only one of the ester groups is sufficient for the degradation of the crosslink. Furthermore, the hydrolysis of the second ester group of the same unit releases N-methyldiethanolamine as a small molecule byproduct, while the acrylate units remain on the polymer.

[0109] The hydrolysis of DXL in phosphate buffered D2O at pH 7.3 maintained at room temperature (22 °C) was 1 monitored by 1H NMR spectroscopy. The results showed that approximately 31% of DXL was hydrolyzed after 1 day at room temperature (22 °C) (Figure 3). A small portion of the monoacrylate product was further hydrolyzed to obtain N-methyldiethanolamine (MDEA) such that the product consisted of 27.2% monoacrylate and 3.4% MDEA. The hydrolysis of one of the ester groups of DXL showed pseudo-first order kinetics, and the rate constant (k) was 0.0151 h -1 −1, and the half-life was approximately 1.91 days (Figure 4).

[0110] The results showed that the hydrolysis of DXL occurred approximately 1.8 times faster than that of poly[N,N-dimethylaminoethyl acrylate] (PDMAEA) at pH 7 with a half-life of approximately 3.5 days. The hydrolysis of DXL polymerized within the particles was expected to occur at a similar rate, and the presence of the comonomer and the polarity within the particles could potentially affect the rate. The structure of DXL or the adjacent functional groups incorporated into the comonomer could also be used to tune the rate of hydrolysis and degradation of the corresponding particles.

[0111] Precipitation polymerization of DXL crosslinked particles DXL was used as a diacrylate crosslinking agent to form degradable cationic particles in an aqueous medium. DXL consists of β - amino ester bonds that have been shown to undergo rapid ester hydrolysis under mild physiological conditions. N,N-(dimethylaminoethyl) acrylate (DMAEA) is an example of a monomer having such a reactive ester that has been studied in the field of degradable polymer materials. It was hypothesized that DXL - crosslinked particles could act as delivery vehicles for therapeutic payloads such as DNA / RNA and proteins due to their cationic charge having a tertiary ammonium group. Then, the DXL - loaded particles could facilitate cell uptake having a net cationic charge and then degrade intracellularly by hydrolysis of the labile ester bonds to release the complexed payload. Degradation decreases the cationic charge on the polymer chain and the soluble polymer can be removed from the body by renal filtration.

[0112] Precipitation polymerization was selected to prepare DXL - crosslinked particles because it does not require the use of surfactants or steric stabilizers as compared to other methods such as emulsion polymerization. DXL was polymerized with HEA as a neutral polar comonomer in a critical solvent system consisting of MEK as a polar good solvent and heptane as a non - polar poor solvent. Since HEA - DXL polymer chains are first formed in the critical solvent, aggregation of the polymer chains forms particle nuclei that capture subsequent polymer chains formed to grow polymer particles. The 70 / 30 HEA / DXL molar feed ratio was selected due to the limited solubility of DXL in the 75 / 25 MEK / heptane solvent system that was found to be optimal for particle formation. This formulation was prepared by both photo - and thermal - initiated free - radical polymerization. The size of the particles ranged from 2.28±0.35 μm (n = 70) for photo - initiated polymerization and 3.60±1.45 μm (n = 70) for thermal - initiated polymerization (Figures 5 and 6 respectively). It should be noted that samples prepared by thermal polymerization were sonicated to separate the particles from larger aggregates.

[0113] The particles formed by photopolymerization were smaller and more narrowly dispersed in diameter compared to those formed by thermal polymerization. The particles formed by photopolymerization were also more sterically stable.

[0114] Since DXL showed limited solubility in a MEK / heptane solvent mixture composition suitable for particle formation, DMAEA was copolymerized with DXL and HEA to increase the tertiary amine content and cationic charge of the particles. This HEA / DMAEA / DXL terpolymer microparticle system (referred to herein as HDD) was attempted at a 1 / 1 / 1 molar feed ratio using an 85 / 15 MEK / heptane solvent mixture and polymerized by thermal and photopolymerization. The HDD particles obtained by thermal and photopolymerization are shown in Figures 7 and 8 respectively.

[0115] The HDD particles prepared by thermal polymerization resulted in a mixture of particles and particle aggregates with a diameter of 1.85 ± 0.47 μm (n = 70). The HDD particles of the same composition prepared by photopolymerization resulted in submicron particles that could not be analyzed by bright-field microscopy.

[0116] Similar to the HD particles, the size of the HDD particles of the same composition was smaller when prepared by thermal polymerization compared to photopolymerization. This difference is also due to the different rates and extents of polymerization, as well as the effect of the polymerization temperature on the dissolving power.

[0117] The HD particles prepared by photopolymerization were selected for further proof-of-concept experiments due to the stability of the resulting particle suspension and the relatively narrow size dispersity of the samples. After purification, the HD particles were resuspended in PBS at pH 7.4 to obtain a mixture of particles and particle aggregates with a diameter of 2.21 ± 0.44 μm (n = 70) (Figure 9).

[0118] Loading of OVA (OVA-FITC) onto HD particles HD particles can bind to proteins and anionic substances such as DNA / RNA due to the net cationic charge from the tertiary ammonium group of DXL in an aqueous medium. Since the HD particles are retained together with the labile ester bonds of DXL, the particles can be degraded and release the bound payload. To demonstrate the ability of the particles to bind to proteins for potential drug delivery applications, the HD particles were exposed to FITC-labeled ovalbumin (OVA-FITC) or Texas Red-labeled ovalbumin (OVA-RED).

[0119] Confocal fluorescence microscopy of unloaded HD particles revealed the presence of autofluorescence detected from particles with maximum intensity in the FITC channel. Since tertiary amine-containing polymers have been shown to exhibit fluorescence characteristics, the fluorescence of the particles may be attributed to DXL. The fluorescence of HD particles in the TRITC channel was weaker than that in the FITC channel under the same acquisition settings (Figs. 10A - 10D).

[0120] Therefore, to minimize interference from HD autofluorescence, OVA-RED was used for the binding study. HD particles loaded with OVA-RED at a 1:1 weight ratio (HD-OVA-RED) were imaged by brightfield and confocal fluorescence microscopy, and the results are shown in Figs. 11A - 11D. When HD particles without OVA-RED were imaged under the same settings, a much weaker signal was present in the TRITC channel (Figs. 12A - 12B), suggesting that the HD particles can actually bind to proteins such as OVA. It was noted that some aggregates of insoluble OVA-RED that were not bound to the HD particles were observed after binding the HD particles to OVA-RED (Fig. 12A). This is likely due to the Texas Red moiety having a reduced solubility in the aqueous medium.

[0121] Cell uptake HD particles loaded with OVA-FITC were exposed to RAW 264.7 macrophage cells as an in vitro model for vaccine delivery. HD-OVA particles were hypothesized to be taken up by immune cells and then undergo intracellular degradation by DXL hydrolysis to release the loaded antigen. Other cationic delivery vehicles based on non-charge-transfer polycations have been shown to be promising in antigen binding, but often pose challenges in antigen release. Non-charge-transfer polycations also present a potential risk of damaging cells and cellular components after delivery due to the toxicity of such polymers.

[0122] After incubating HD-OVA-FITC particles with RAW 264.7 cells for 4 hours, excess particles in the solution were removed by washing twice with PBS. The cells were then imaged by bright-field microscopy (Figures 13A - C). Figure 13A shows that the morphology of the cells treated with HD-OVA-FITC particles was similar to that of untreated cells incubated in DMEM (Figure 13C). Figure 13A also shows that the fraction of HD-OVA-FITC particles appeared to aggregate to form larger clusters. As a negative control, cells were incubated with 10% v / v DMSO in DMEM, which showed round cell morphology and signs of apoptosis (Figure 13B).

[0123] Since autofluorescence from both HD particles and cells can contribute to the signal in the FITC channel, images of cells alone (Figures 14A - 14D) and cells treated with unloaded HD particles (Figures 15A - 15D) were collected. In both cases, no fluorescence detectable from the cells was seen, except for some particle aggregates on the surface of single cells in Figure 15.

[0124] Next, the cells were imaged by confocal fluorescence microscopy to check for the internalization of HD-OVA-FITC particles. Images of the cells stained with NucBlue nuclear stain after treatment with HD-OVA-FITC are shown in FIGS. 16A - 16D. Fluorescence in the FITC channel was observed from the cells as both diffuse and punctate regions within the cells, suggesting that OVA-FITC was internalized. The co-localization of HD-OVA-FITC particles around the cell nucleus was observed by confocal microscopy of the combined FITC and DAPI channels (FIG. 17). This suggested that OVA-FITC had successfully invaded the macrophages. These results demonstrate that macrophages preferentially take up cationic particles due to their interaction with the negatively charged cell membrane.

[0125] The HD-OVA-FITC particles were evaluated for their cytotoxicity with RAW 264.7 cells by using the MTS proliferation assay. As controls, the cells were incubated with serum-free DMEM, a 0.1 wt% HD particle suspension prepared in serum-free DMEM, and 10% v / v DMSO in serum-free DMEM (all supplemented with 1% penicillin-streptomycin). After 4 hours of incubation, the mean viability of RAW 264.7 cells was 106 ± 31%, 100 ± 22%, 117 ± 23%, and 64 ± 12%, respectively (FIG. 18).

[0126] The decrease in viability of RAW 264.7 cells in 10% v / v DMSO compared to any sample containing particles (0.1 wt% HD-OVA-FITC, 0.1 wt% HD) was statistically significant (p < 0.05), but the difference in viability of RAW 264.7 cells in serum-free DMEM compared to any sample containing particles was not statistically significant (p > 0.05). The results suggested that HD particles with and without OVA loading at 0.1 wt% were not significantly cytotoxic as the viability of the treated cells was equivalent to that of the control cells in serum-free medium.

Claims

1. Polycation carrier particles obtained by polymerizing an acrylate monomer with one or more hydrophilic monomers to form crosslinks, wherein the polycation carrier particles have a net positive charge, and the acrylate monomer is of formula I or formula V: 【Chemistry 1】 【Chemistry 2】 (In the formula, R and R'' are independently hydrogen and C) １ -C １４ Linear or branched alkyl, C ３ -C ８ A cycloalkyl group, or a 5-10 membered aryl or heteroaryl ring, optionally terminated with a polymerizable group that can crosslink with one or more hydrophilic monomers, preferably an acrylate group. R' is C ２ -C １４ Linear or branched alkyl, C ３ -C ８ It is a cycloalkyl or a 5-10 membered aryl or heteroaryl ring, where R' is terminated with at least one polymerizable group that can crosslink with the one or more hydrophilic monomers, preferably an acrylate group. Alkyl, cycloalkyl, aryl, and heteroaryl groups may be substituted or interrupted by one or more oxygen, sulfur, or nitrogen atoms. R ２ is C ２ or C ３ where C ３ is linear or cyclic), a polycation carrier particle selected from.

2. The polycation carrier particle according to claim 1, wherein the polymerizable group is selected from an acrylate group, a methacrylate group, a (meth)acrylamide group, or a styrene group.

3. The acrylate monomer of formula I is Formula II: 【Transformation 3】 (In the formula, R １ is hydrogen or C １ -C １４ The linear, branched, or cyclic alkyl group may be substituted, interrupted by one or more oxygen, sulfur, or nitrogen atoms, or terminated with an acrylate group. R ２ C ２ or C ３ And here, C ３ It is linear or cyclic, R ３ C ２ -C １４ (It is a linear or branched alkyl group, which may be substituted and interrupted by one or more oxygen, sulfur, or nitrogen atoms.) Polycation carrier particles according to claim 1 or 2, wherein the polycation carrier particles are multiacrylate monomers.

4. The multiacrylate monomer is Formula III: 【Chemistry 4】 (In the formula, R １ and R ２ R is defined as in claim 3, ４ ha-CH ２ -CH ２ -O- or -CH ２ -CH ２ -CH ２ -O- and R ４ terminal CH ２ The polycation carrier particle according to claim 3, wherein the group is bonded to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3, or 4.

5. The multiacrylate monomer is Formula IV: 【Transformation 5】 (In the formula, R １ and R ２ R is defined as in claim 3, １ ' is R １ It has the same definition as R ２ ' is C ２ -C ３ And here C ３ The polycation carrier particle according to claim 3, wherein m is linear or cyclic, and m is an integer selected from 0, 1, 2, 3, or 4.

6. The acrylate monomer of formula V is Formula VI: 【Transformation 6】 (In the formula, R １ and R ５ These are, independently, hydrogen or C １ -C １４ The linear, branched, or cyclic alkyl group may be substituted, interrupted by one or more oxygen, sulfur, or nitrogen atoms, or terminated with an acrylate group. R ２ C ２ or C ３ And here, C ３ It is linear or cyclic, R ３ C ２ -C １４ The polycation carrier particle according to claim 1, which is a linear or branched alkyl multiacrylate monomer (which may be substituted and interrupted by one or more oxygen, sulfur, or nitrogen atoms).

7. The multiacrylate monomer is Formula VII: 【Transformation 7】 (In the formula, R １ and R ５ R is defined as in claim 6, ４ ha-CH ２ -CH ２ -O- or -CH ２ -CH ２ -CH ２ -O- and R ４ terminal CH ２ The polycation carrier particle according to claim 6, wherein the group is bonded to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3, or 4.

8. The polycation carrier particle according to claim 5, wherein one or more of the nitrogen atoms are protonated.

9. The multiacrylate monomer is Formula VIII: 【Transformation 8】 (In the formula, R １ and R ５ R is defined as in claim 6, ２ is C ２ or C ３ And here, C ３ The chain is linear or ring-shaped, m is an integer selected from 0, 1, 2, 3, or 4, and R １ ', R ２ 'and R ５ ' represents R １ , R ２ and R ５ The polycation carrier particle according to claim 8, which independently shares the definition of

10. The acrylate monomer is 【Chemistry 9】 Polycation carrier particles according to claim 1, selected from the above.

11. The polycation carrier particle according to claim 1, wherein the multiacrylate monomer is methyldiethanolamine diacrylate (DXL).

12. The hydrophilic monomer is Formula IX: 【Chemistry 10】 (In the formula, R ６ ha-O-(CH ２ ) ｐ -Y, -NJK, or -NJ ２ K ＋ Here, p is an integer selected from 2, 3, or 4, and Y is OH, COOH, NJ. ２ NJ ３ ＋ Or ONJ ２ Defined as such, each J is independently defined as H, methyl, or ethyl, and K is ethyl, propyl, or butyl, which may be branched or substituted, R ７ The polycation carrier particle according to claim 1, wherein ( is hydrogen or methyl).

13. The hydrophilic monomer is Formula X: 【Chemistry 11】 (In the formula, R ８ is C ２ -C ４ The polycation carrier particle according to claim 1, wherein X is a linear alkyl group, and X is selected from hydroxyl, carboxyl, tertiary amine, quaternary ammonium or amide, and the amine may be substituted with a methyl or ethyl group.

14. The polycation carrier particle according to claim 1, wherein one or more hydrophilic monomers are 2-hydroxyethyl acrylate (HEA) and / or N,N-(dimethylamino)ethyl acrylate (DMAEA).

15. The polycation carrier particles according to claim 1, wherein the polycation carrier particles have a size of 200 nm to 5 μm.

16. The polycation carrier particle according to claim 1, wherein the mole percent ratio of the acrylate monomer to the one or more hydrophilic monomers is 15 / 85 to 50 / 50.

17. The polycation carrier particle according to claim 1, further comprising a cargo material composited with the polycation carrier particle.

18. A complex comprising polycation carrier particles complexed with a cargo molecule, wherein the polycation carrier particles are adapted to release the cargo molecule into a cell, are degradable under physiological conditions, have charge shift properties, and are obtained by polymerizing one or more hydrophilic monomers with an acrylate monomer of formula I or formula V as defined in claim 1.

19. A method for producing polycation carrier particles as defined in claim 1, wherein the method is: To provide an acrylate monomer as defined in claim 1 and one or more hydrophilic monomers as defined in claim 1, A method comprising crosslinking the acrylate monomer with one or more hydrophilic monomers.

20. The method according to claim 19, wherein the crosslinking step includes one of precipitation polymerization, microemulsion polymerization, dispersion polymerization, reverse suspension polymerization, or reverse emulsion polymerization.

21. The method according to any one of claims 19 or 20, wherein only one hydrophilic monomer is provided, and the molar percentage ratio of the acrylate monomer to the hydrophilic monomer is 30 ± 15 / 70 ± 15.

22. The method according to claim 19, wherein two hydrophilic monomers are provided, and the molar percentage ratio of the acrylate monomer to the two hydrophilic monomers is 33±10 / 33±10 / 33±10.

23. The method according to claim 21, wherein the one hydrophilic monomer is HEA and the acrylate monomer is DXL.

24. The method according to claim 22, wherein the two hydrophilic monomers are HEA and DMAEA, and the acrylate monomer is DXL.

25. The method according to claim 19, further comprising providing a drug to be encapsulated, and crosslinking the acrylate monomer with one or more hydrophilic monomers in a medium containing the drug.

26. A method for producing a composite of cargo material and polycation carrier particles, To produce polycation carrier particles by the method of claim 19, A method for obtaining the composite, comprising: exposing the polycation carrier particles to a solution containing the cargo substance, thereby compositering the negatively charged cargo substance with the positively charged polycation carrier particles.

27. A method for delivering a vaccine to a target requiring it, comprising administering the polycation carrier particles described in claim 1 to the target, wherein the polycation carrier particles are complexed with a vaccine agent or contain a vaccine composition.

28. A method for inducing an in vivo immune response, comprising administering to a subject in need of such response a polycation carrier particle according to claim 1, which is complexed with an antigen or a nucleic acid encoding an antigen, or encapsulating a vaccine composition.

29. A method for delivering nucleic acids for in vivo protein expression, comprising administering the polycation carrier particles described in claim 1 to a target, wherein the polycation carrier particles are complexed with a nucleic acid encoding a protein.

30. A vaccine composition comprising polycation carrier particles according to claim 1, which are complexed with an antigen or nucleic acid encoding an antigen, and a pharmaceutically acceptable carrier or adjuvant.

31. The vaccine composition according to claim 30, wherein the nucleic acid is mRNA.

32. The complex according to claim 18, wherein the cargo molecule is selected from nucleic acids, peptides, and proteins.

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