Polycationic carrier particles

Abstract

A polycationic carrier particle obtained by polymerization of a polyacrylate monomer with one or more hydrophilic monomers to form a cross-link. The polycationic carrier particles have a net positive charge. The multi-acrylate monomer and the hydrophilic monomer are as defined herein.

Description

[0001] Citation of Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 350,275, filed on June 8, 2022, the contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of carrier particles for therapeutic agents, vaccines, and the like, and more particularly, to polycationic particles and methods and uses thereof and their preparation. Background Art

[0004] In vivo delivery of proteins and genetic materials in the form of polynucleic acids (such as mRNA, DNA and siRNA) has a wide range of therapeutic application potentials from gene knockout and editing to vaccines based on mRNA / DNA and proteins. The success of applying proteins and polynucleotides to in vivo treatment depends on the ability of the required genetic load to be delivered to target cells intracellularly. The delivery of such proteins and large polynucleic acids requires the use of delivery vectors to provide stability for the load during circulation, and usually promotes cellular uptake by endocytosis.

[0005] Many synthetic material-based approaches have been used to produce delivery vehicles. Most notably, lipid nanoparticle-based delivery of mRNA for COVID-19, developed by Pfizer and Moderna, has achieved widespread clinical success. Polycations have also been explored as synthetic materials for nucleic acid complexation and protein delivery. Proteins are protected by polycationic carrier particles prior to endocytosis and released from endosomes via the proton sponge effect. Benefits of synthetic polycations over lipid nanoparticles include ease of production, stability, low cost, and synthetic control over polymer architecture and composition (allowing for tunability of features such as circulation time, release kinetics, and targeted cell delivery).

[0006] For example, a significant challenge in using polycations in delivering nucleic acid materials is the general cytotoxicity associated with the polycations. After cellular uptake, dissociation of the complex between the polycation and the encapsulated material (e.g., genetic payload) results in the release of the encapsulated material as well as the potentially cytotoxic polycation. Therefore, it is desirable to improve polycation carrier particles so that once the particles dissociate within the cell to release the encapsulated material, the cytotoxicity of the polycation will be reduced. Summary of the invention

[0007] In one aspect, a polycationic carrier particle is provided which is obtained by polymerizing an acrylate monomer with one or more hydrophilic monomers to form crosslinks, wherein the polycationic carrier particle has a net positive charge, and wherein the acrylate monomer is selected from Formula I or Formula V.

[0008]

[0009] R and R" are each independently hydrogen, C1-C 14 A straight or branched alkyl group, a C3-C8 cycloalkyl group or a 5 to 10 membered aryl or heteroaryl ring, which is optionally terminated by a polymerizable group capable of crosslinking with one or more hydrophilic monomers, preferably an acrylate group. 14 A straight or branched alkyl group, a C3-C8 cycloalkyl group or a 5 to 10-membered aryl or heteroaryl ring, and R' is terminated by at least one polymerizable group capable of crosslinking with one or more hydrophilic monomers, preferably an acrylate group. Alkyl, cycloalkyl, aryl and heteroaryl groups may be optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms. Furthermore, R2 is C2 or C3, and C3 is straight or cyclic.

[0010] In some embodiments, the polymerizable group is selected from an acrylate group, a methacrylate group, a (meth)acrylamide group, or a styrene group.

[0011] In some embodiments, the acrylate monomer of formula I is a multiacrylate monomer of formula II

[0012]

[0013] R1 is hydrogen or C1-C 14 A linear, branched or cyclic alkyl group, which is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and optionally terminated with an acrylate group, R2 is C2 or C3, and C3 is linear or cyclic, R3 is C2-C 14 Straight or branched chain alkyl, which is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

[0014] In some embodiments, the multiacrylate monomer has Formula III.

[0015]

[0016] R1 and R2 are as previously defined, R4 is -CH2-CH2-O- or -CH2-CH2-CH2-O-, and the terminal CH2 group of R4 is connected to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3 or 4.

[0017] In some embodiments, the multiacrylate monomer has Formula IV.

[0018]

[0019] R1 and R2 are as previously defined, 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 of Formula IV are protonated.

[0020] In some embodiments, the acrylate monomer of Formula V is a multiacrylate monomer of Formula VI.

[0021]

[0022] R1 and R5 are independently hydrogen or C1-C 14 A linear, branched or cyclic alkyl group, which is optionally substituted and 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 Straight or branched chain alkyl, which is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

[0023] In some embodiments, the multiacrylate monomer has Formula VII.

[0024]

[0025] R1 and R5 are as defined above, R4 is -CH2-CH 2- O- or -CH2-CH2-CH2-O-, and the terminal CH2 group of R4 is connected to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3 or 4.

[0026] In some embodiments, the multiacrylate monomer has Formula VIII.

[0027]

[0028] R1 and R5 are as previously defined, 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' independently share the definitions of R1, R2 and R5, respectively.

[0029] In some embodiments, the acrylate monomer is selected from:

[0030]

[0031] In some embodiments, the multiacrylate monomer is methyldiethanolamine diacrylate (DXL).

[0032] In some embodiments, the hydrophilic monomer has Formula IX.

[0033]

[0034] R6 is -O-(CH2) p -Y, -NJK, or -NJ2K + , p is an integer selected from 2, 3 or 4, and Y is defined as OH, COOH, NJ2, NJ3 + or 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.

[0035] In some embodiments, the hydrophilic monomer has formula X.

[0036]

[0037] R8 is C 2- C4 straight chain alkyl, X is selected from hydroxyl, carboxyl, tertiary amine, quaternary ammonium or amide, and the amine is optionally substituted by methyl or ethyl.

[0038] In some embodiments, the one or more hydrophilic monomers are 2-hydroxyethyl acrylate (HEA) and / or N,N-(dimethylamino)ethyl acrylate (DMAEA).

[0039] In some embodiments, the polycationic carrier particles have a size of 200 nm to 5 μm.

[0040] In some embodiments, the mole percentage of acrylate monomer to one or more hydrophilic monomers is 15 / 85 to 50 / 50.

[0041] In some embodiments, the polycationic carrier particle further comprises a load material complexed with the polycationic carrier particle.

[0042] In one aspect, a complex is provided, comprising a polycationic carrier particle complexed with a load molecule, the polycationic carrier particle being suitable for releasing the load molecule intracellularly and being degradable under physiological conditions, and wherein the polycationic carrier particle has charge transfer properties and is obtained by polymerizing one or more hydrophilic monomers with an acrylate monomer of formula I or formula V as defined herein. In one embodiment, the load molecule is selected from nucleic acids, peptides and proteins.

[0043] In one aspect, there is provided a method of preparing a polycationic carrier particle 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.

[0044] In some embodiments, the cross-linking step comprises performing one of precipitation polymerization, miniemulsion polymerization, dispersion polymerization, inverse suspension polymerization, or inverse emulsion polymerization.

[0045] In some embodiments, only one hydrophilic monomer is provided, and the molar percentage of the acrylate monomer to the hydrophilic monomer is 30±15 / 70±15. In one example, the one hydrophilic monomer is HEA, and the acrylate monomer is DXL.

[0046] In some embodiments, two hydrophilic monomers are provided, and the molar percentage of the 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.

[0047] In some embodiments, the method further comprises providing an agent to be encapsulated and cross-linking the acrylate monomer with the one or more hydrophilic monomers in a medium comprising the agent.

[0048] In another aspect, a method for producing a complex of a load material and a polycationic carrier particle is provided, the method comprising: producing the polycationic carrier particle by the method as defined herein; and complexing the negatively charged load material to the positively charged polycationic carrier particle by exposing the polycationic carrier particle to a solution containing the load material, thereby obtaining the complex.

[0049] In one aspect, a method of delivering a vaccine to a subject in need thereof is provided, the method comprising administering to the subject a polycationic carrier particle as defined herein, wherein the polycationic carrier particle is complexed with a vaccine agent or encapsulates a vaccine composition.

[0050] In one aspect, there is provided a method of inducing an immune response in vivo comprising administering to a subject in need thereof a polycationic carrier particle as defined herein, complexed with an antigen or a nucleic acid encoding an antigen or encapsulating a vaccine composition.

[0051] In one aspect, a method of nucleic acid delivery for in vivo protein expression is provided, the method comprising administering to a subject a polycationic carrier particle as defined herein, wherein the polycationic carrier particle is complexed with a nucleic acid encoding the protein.

[0052] In one aspect, a vaccine composition is provided comprising a polycationic carrier particle as defined herein, complexed with an antigen or a nucleic acid encoding an antigen and a pharmaceutically acceptable carrier or adjuvant. In one embodiment, the nucleic acid is mRNA.

[0053] After reading this disclosure, many additional features and combinations thereof related to improvements of the present invention will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 is the product of N-methyldiethanolamine diacrylate [(methylazanediyl)bis(ethane-2,1-diyl) diacrylate] (DXL) in CDCl3 recorded at 600 MHz 1 H nuclear magnetic resonance (NMR) spectroscopy.

[0055] Figure 2 The time course of DXL in 100% phosphate-buffered D2O at pH 7.30 and room temperature (22°C) is shown. 1 H NMR spectroscopy.

[0056] Figure 3 is a graph showing the ester hydrolysis of DXL over time, more specifically, the ester hydrolysis of DXL monomer in 100 mM phosphate buffered D2O at pH 7.30, room temperature (22°C). The percent (%) hydrolysis data represents the hydrolysis of one of the ester groups of DXL.

[0057] Figure 4 is a graph showing a pseudo first order plot of the ester hydrolysis of DXL monomer in 100 mM phosphate buffered D2O at pH 7.30, room temperature (22° C.) The data represent the hydrolysis of one of the ester groups of DXL.

[0058] Figure 5 is a bright field microscopy image at 50X objective and 5 μm scale bar showing the polymerization mixture of 70 / 30 molar feed ratio of 2-hydroxyethyl acrylate (HEA) / DXL in 75 / 25M methyl ethyl ketone (MEK) / heptane solvent mixture after 20-24 hours of photopolymerization.

[0059] Figure 6 is a bright field microscopy image at 50X objective and 5 μm scale bar showing a polymerization mixture of 70 / 30 molar feed ratio of HEA / DXL in 75 / 25 MEK / heptane solvent mixture after thermal polymerization at 70°C for 5-8 hours followed by purification and sonication in 1,4-dioxane.

[0060] Figure 7 is a bright field microscopy image at 50X objective and 10 μm scale bar showing a 1 / 1 / 1 molar feed ratio suspension of DXL / N,N-(dimethylamino)ethyl acrylate (DMAEA) / HEA particles prepared in a 85 / 15 MEK / heptane solvent mixture thermal polymerization after purification and sonication in 1,4-dioxane.

[0061] Figure 8 is a bright field microscopy image at 50X objective and 5 μm scale bar showing a polymerized mixture of 1 / 1 / 1 DXL / DMAEA / HEA particles in 85 / 15 MEK / heptane solvent mixture after photopolymerization.

[0062] Fig. 9 is a bright field microscopy image at 50X objective and 10 μm scale bar showing a polymerization mixture of 70 / 30 molar feed ratio of HEA / DXL in 75 / 25 MEK / heptane solvent mixture after 20-24 hours of photopolymerization, washing with 1,4-dioxane, and sonication in phosphate buffered saline (PBS) at pH 7.4.

[0063] Fig. 10A Confocal microscopy images of particles obtained with 70 mol% HEA and 30 mol% DXL in 75 / 25 MEK / heptane solvent mixture at 1 g total monomer loading, 1 mol% azobisisobutyronitrile (AIBN) as free radical initiator for photolysis, washed twice with phosphate buffered saline, imaged on confocal with 20X objective (scale bar indicates 10 μm), and merged channels are shown: bright field, fluorescein isothiocyanate (FITC), and tetramethylrhodamine isothiocyanate (TRITC).

[0064] Fig. 10B are confocal microscopy images of 70:30 HEA / DXL particles (imaged with a 20X objective (scale bar indicates 10 μm) and showing the bright field channel).

[0065] Fig. 10C are confocal microscopy images of 70:30 HEA / DXL particles (imaged with a 20X objective (scale bar indicates 10 μm) and showing the FITC channel).

[0066] Fig. 10D are confocal microscopy images of 70:30 HEA / DXL particles (imaged with a 20X objective (scale bar indicates 10 μm) and showing the TRITC channel).

[0067] Fig.11A Texas Red TM Confocal microscopy images of 70:30 HEA / DXL particles (washed twice with PBS) combined with conjugate (OVA-RED) in a 1:1 weight ratio (imaged on confocal with a 20X objective (where the scale bar indicates 25 μm), showing merged bright field FITC and TRITC channels).

[0068] Fig. 11Bare confocal microscopy images (imaged on a confocal with a 20X objective (where the scale bar indicates 25 μm), bright field channel shown) of 70:30 HEA / DXL particles (washed twice with PBS) combined with OVA-RED at a 1:1 weight ratio.

[0069] Fig. 11C are confocal microscopy images (imaged on confocal with 20X objective (where scale bar indicates 25 μm), FITC channel shown) of 70:30 HEA / DXL particles (washed twice with PBS) combined with OVA-RED at a 1:1 weight ratio.

[0070] Fig.11D are confocal microscopy images (imaged on confocal with 20X objective (where scale bar indicates 25 μm), TRITC channel shown) of 70:30 HEA / DXL particles (washed twice with PBS) combined with OVA-RED at a 1:1 weight ratio.

[0071] Fig. 12A is a confocal microscopy image of 70:30 HEA / DXL particles (washed twice with PBS) combined with OVA-RED at a 1:1 weight ratio (imaged on a confocal with a 20X objective (where the scale bar indicates 25 μm)

[0072] Fig. 12B are confocal microscopy images of 70:30 HEA / DXL particles without OVA-RED (washed twice with PBS) (imaged on a confocal with a 20X objective (where the scale bar indicates 25 μm (B)).

[0073] Fig.13A Figure 1 is a bright field microscopy image of RAW 264.7 cells in a glass bottom 96-well plate (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin-streptomycin. The cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged by bright field microscopy using a 20X objective (where the scale bar indicates 50 μm).

[0074] Fig. 13BFigure 1 is a bright field microscopy image of RAW 264.7 cells in a glass bottom 96-well plate (Cellvis) after incubation in serum-free DMEM with 10% v / v dimethyl sulfoxide (DMSO) for 4 hours at 37°C in a humidified atmosphere with 5% CO. The cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged by bright field microscopy with a 20X objective (where the scale bar indicates 50 μm).

[0075] Fig. 13C is a bright field microscopy image of RAW 264.7 cells in a glass bottom 96-well plate (Cellvis) after incubation in serum-free DMEM for 4 hours at 37°C in a humidified atmosphere with 5% CO. The cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged by bright field microscopy with a 20X objective (where the scale bar indicates 50 μm).

[0076] Fig.14A Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 h with serum-free DMEM supplemented with 1% penicillin-streptomycin at 37° C. in a humidified atmosphere with 5% CO 2. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective (where the scale bar indicates 10 μm, merged bright field, FITC, and TRITC channels are shown).

[0077] Fig. 14B Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 h with serum-free DMEM supplemented with 1% penicillin-streptomycin at 37° C. in a humidified atmosphere with 5% CO 2. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective (where the scale bar indicates 10 μm, bright field channel shown).

[0078] Fig. 14C Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 hours with serum-free DMEM supplemented with 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective (where the scale bar indicates 10 μm, FITC channel is shown)

[0079] Fig.14D Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 h with serum-free DMEM supplemented with 1% penicillin-streptomycin at 37° C. in a humidified atmosphere with 5% CO 2. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective (where the scale bar indicates 10 μm, TRITC channel is shown).

[0080] Fig.15A Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling, scale bar indicates 10 μm, and merged bright field, FITC, and TRITC channels are shown.

[0081] Fig. 15B Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 hours at 37°C with a 0.1 wt% HD particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin in a humidified atmosphere with 5% CO2. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling, scale bar indicates 10 μm, and bright field channel is shown.

[0082] Fig. 15C Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling, scale bar indicates 10 μm, and FITC channel is shown.

[0083] Fig.15DConfocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 hours at 37°C with a 0.1 wt% HD particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin in a humidified atmosphere with 5% CO2. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling, scale bar indicates 10 μm, and TRITC channel is shown.

[0084] Fig.16A Confocal microscopy images of RAW 264.7 cells in a glass-bottom 96-well plate (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin. The cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling. The scale bar indicates 10 μm. Merged bright field, FITC, and 4',6-diamidino-2-phenylindole (DAPI) channels are shown.

[0085] Fig. 16B Confocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling. The scale bar indicates 10 μm. The merged bright field channel is shown.

[0086] Fig. 16CConfocal microscopy images of RAW 264.7 cells in glass-bottom 96-well plates (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling. The scale bar indicates 10 μm. The FITC channel is shown.

[0087] Fig.16D Confocal microscopy images of RAW 264.7 cells in a glass-bottom 96-well plate (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling. The scale bar indicates 10 μm. The DAPI channel is shown.

[0088] Fig.17 Confocal microscopy images of RAW 264.7 cells in a glass-bottom 96-well plate (Cellvis) after incubation for 4 hours at 37°C in a humidified atmosphere with 5% CO2 with a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin. Cells were washed twice with PBS, fixed by 4% v / v paraformaldehyde in sterile water, stored in PBS, and imaged on a confocal with a 60X oil immersion objective and Nyquist XY sampling. The scale bar indicates 10 μm. The merged FITC and DAPI channels are shown.

[0089] Fig.18is a bar graph showing the viability results of the MTS cytotoxicity assay reported as mean ± standard deviation in the case of the RAW 264.7 cell line. The "control" results were obtained from RAW 264.7 cells in serum-free DMEM supplemented with 1% penicillin-streptomycin. The "HD-OVA-ITC 0.1 wt %, "HD 0.1 wt %", and "DMSO 10% v / v" results were obtained from RAW 264.7 cells in 0.1 wt % HD-OVA-FITC particle suspension prepared in serum-free DMEM, 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), respectively. DETAILED DESCRIPTION

[0090] The present disclosure relates to carrier particles made from "charge transfer" polycations. These charge transfer polycations are materials containing an initial high cationic charge density that allows for complexation with materials associated with the carrier particle (e.g., vaccines, proteins, and / or nucleic acids). The carrier particles can then be subjected to cellular uptake, where a charge transfer mechanism will occur, typically through hydrolysis, thereby reducing the overall cationic charge density to improve dissociation and release of the composite material (i.e., the load or payload of the polycationic carrier particle).

[0091] Traditionally, charge-transfer polycations for cellular delivery have focused on the use of soluble polymers in branched or linear polymer architectures. One limitation of soluble polycations is that the formation of polymers with nucleic acids is a difficult process to control particle morphology, which strongly affects particle uptake.

[0092] In vivo drug delivery by polymer particles requires final degradation to cell-compatible byproducts under physiological conditions. For cross-linked materials, this requires a degradable cross-linking agent. Generally, cross-linking agents containing disulfide, acetal and ketal have been used as biodegradable functional groups, which convert cross-linked polymer particles into soluble polymers when degraded. Although these functional groups are biodegradable, there are also restrictive problems associated with them. Acetals and ketals are functional groups that can undergo acid-catalyzed hydrolysis, and alkaline pH values ​​in aqueous solutions are required before in vivo application to ensure the stability of the material. When carrier particles with acetal and ketone functional groups are endocytosed, the acidic pH of endosomes and lysosomes can trigger the release of the contents of the particles before the particles reach the cytoplasm or nucleus. Therefore, ketals and acetals may be less desirable functional groups for cell delivery. In addition, acetal and ketal cross-linking agents are generally relatively hydrophobic, so they are prone to problems of water solubility and protein binding through hydrophobic interactions. Disulfide is another example of a functional group traditionally used in carrier particles. When disulfides are cleaved or degraded, they generate free thiol groups, which are reactive and may be involved in undesirable side reactions (e.g., with proteins, DNA, etc.) Therefore, disulfides may also be less desirable as functional groups in carrier particles.

[0093] The present disclosure provides a polycationic carrier particle that overcomes the above-noted disadvantages while providing the advantages of improved biocompatibility, reduced cytotoxicity and biodegradability of the polycationic carrier particle and the degradation byproducts produced. The polycationic carrier particles of the present disclosure can be degraded by hydrolysis to form acrylic acid units on the polymer, which are non-toxic and commonly used in biocompatible materials. The degradation products produced are hydrolyzed small molecules that are biocompatible and can be cleared out of the body over time by renal clearance. In some embodiments, the hydrolyzed small molecules are physiologically inert and preferably do not contain acrylic acid groups. The chemical structure of the hydrolyzed small molecules is generally similar to that of known food additives, such as dimethyl ethanol. In order to achieve these improvements, the polycationic carrier particles of the present disclosure are formed by acrylate monomers and one or more hydrophilic comonomers. The crosslinking agent of the present disclosure that forms the polycationic carrier particles combines the improved performance of charge transfer materials for polycation-based drug delivery with the biodegradability of the crosslinking agent. In one embodiment, polycation carrier particles can be synthesized by self-stabilizing precipitation polymerization, which exploits a balance of polymer solubility and crosslinker loading to produce narrowly dispersed charge transfer polycation carrier particles.

[0094] As used herein, the term "acrylate" refers to a compound having one or more acrylate groups. In some embodiments, the acrylate monomer is a "multi-acrylate" 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).

[0095] As used herein, the term "polycationic" refers to a polymer having a net positive charge formed from an acrylate monomer as described herein and one or more hydrophilic comonomers.

[0096] As used herein, the term "degradable" means degradation occurs under physiological conditions, wherein the time range is about 2 hours to 2 weeks, preferably 8 hours to 48 hours. The degradation may include hydrolysis of acrylate and acrylic acid groups and decomposition of the polycationic polymer into small molecules.

[0097] As used herein, the term "optionally substituted" can be defined as having one or more substitutions selected from halogens (e.g., Cl, Br, F, and I), oxides, amines, amides, carboxyls, alkoxys, hydroxyls, alcohols, esters, ethers, nitro, nitrile, alkyls (e.g., C1-C6), aryls (e.g., phenyls), acrylamides, and other known biocompatible substituents. In some embodiments, the substituents can exclude phosphates, sulfates, and other groups that counteract cationic charge.

[0098] In some embodiments, the acrylate monomers disclosed herein have formula I

[0099]

[0100] R is hydrogen, C1-C 14 A straight chain or branched alkyl group, a C3-C8 cycloalkyl group, or a 5- to 10-membered aryl or heteroaryl ring. 14 Straight or branched alkyl, C3-C9 cycloalkyl or 5 to 10 yuan of aryl or heteroaryl rings, which are end-capped with at least one polymerizable group that can be cross-linked with one or more hydrophilic monomers, preferably acrylate groups. Alkyl, cycloalkyl, aryl and heteroaryl can be optionally substituted and optionally interrupted by one or more oxygen, sulphur or nitrogen atoms. The polymerizable group can be selected from acrylate groups, methacrylate groups, (methyl) acrylamide groups or styrene groups. In some embodiments, R and R' can be connected and form a nitrogen-containing heterocycloalkyl combined with R and R' shown in Formula I. For example, a C3-C8 heterocycloalkyl optionally substituted can be formed.

[0101] In some embodiments, R is defined as 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. R can be linear or branched, optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and optionally terminated by 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 is C2, and is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and is terminated by at least one polymerizable group.

[0102] The acrylate monomer of the present disclosure is a crosslinker, which means that it has at least two polymerizable groups, including the acrylate group attached to R2 in the formula shown herein. A monomer having a single polymerizable group will not allow the formation of polymer particles, and will not allow the crosslinking required to form such particles, but will instead produce a linear polymer. In a preferred embodiment shown below, the acrylate monomer is a multi-acrylate monomer having multiple acrylate groups, thereby allowing for improved crosslinking, as having more groups that can react increases the efficiency of the crosslinking.

[0103] 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. As shown in Formula I, the acrylate monomer of the present disclosure has an acrylate group adjacent to an amine, wherein the spacer between the two groups is R2. This feature of the acrylate monomer allows the acrylate monomer to be polymerized into polycationic carrier particles by a reaction of the acrylate group initiated by the amine.

[0104] The amine plays a direct inductive and nucleophilic role in activating the ester for hydrolysis. It also helps to attract water and hydroxide ions, which react with the ester during hydrolysis and may further activate the ester carbonyl group by hydrogen bonding.

[0105] In some embodiments, the acrylate monomer is a multiacrylate monomer of formula II

[0106]

[0107] R1 is hydrogen or C1-C 14A linear, branched or cyclic alkyl group, which is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and optionally terminated by an acrylate group. R2 is C2 or C3, wherein C3 is linear or cyclic, preferably linear. R3 is C2-C 14 Straight or branched chain alkyl, which is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

[0108] In some embodiments, R1 is defined as 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 can be linear or branched, optionally substituted and 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 is C2 and is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

[0109] In some embodiments, the multi-acrylate monomer has Formula III

[0110]

[0111] R1 and R2 are as previously defined. R4 is -CH2-CH2-O- or -CH2-CH2-CH2-O-, and the terminal CH2 group of R4 is connected to the oxide of the terminal acrylate group. And, n is an integer selected from 1, 2, 3 or 4.

[0112] In some embodiments, the multiacrylate monomer has Formula IV

[0113]

[0114] R1 and R2 are as previously defined. 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-, -CH 2- CH2-CH2- or cyclopropyl, preferably -CH2-CH2 or -CH2-CH2-CH2-. In some embodiments, R2' is unsubstituted.

[0115] In some embodiments, the acrylate monomer has Formula V:

[0116]

[0117] R, R' and R2 are as previously defined. R" independently follows the same definition as R. In some embodiments, two or more of R, R' and R" can be linked and form a nitrogen-containing heterocycloalkyl group combined with R, R' and R" as shown in Formula V. For example, an optionally substituted C3-C8 heterocycloalkyl group can be formed.

[0118] In some embodiments, the acrylate monomer of Formula V can be a multi-acrylate monomer. In some embodiments, the multi-acrylate monomer has Formula VI.

[0119]

[0120] R1, R2 and R3 are as previously defined. R5 independently follows the definition of R1 provided above.

[0121] In some embodiments, the multiacrylate monomer has Formula VII

[0122]

[0123] wherein R1, R4, R5 and n are as previously defined.

[0124] In some embodiments, the multiacrylate monomer of formula IV may have one or both of the nitrogen atoms protonated and attached to groups R5 and R5' (both independently following the definition of R1 provided previously). For example, the multiacrylate monomer of formula IV may have both nitrogens protonated as shown below in formula VIII.

[0125]

[0126] R1, R1′, R2, R2′, R5, R5′ and m are as previously defined.

[0127] In a preferred embodiment, the acrylate monomer is selected from:

[0128]

[0129] In one embodiment, the acrylate monomer is N-methyldiethanolamine diacrylate, referred to as (DXL), having the formula:

[0130]

[0131] The acrylate monomer can be a cationic charge transfer monomer that contributes to the net positive charge and charge transfer properties of the generated polycation. The acrylate monomer is crosslinked or polymerized with one or more hydrophilic monomers to obtain polycationic carrier particles. The acrylate monomer is crosslinked with one or more hydrophilic monomers to reduce the crosslink density of the generated 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).

[0132] In some embodiments, the hydrophilic monomer is a neutral polar monomer. In some embodiments, the hydrophilic monomer has a molecular weight less than 500g / mol, less than 400g / mol or less than 300g / mol. Smaller molecules are advantageous because 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 cationic charge, and therefore contributes to the total charge of the polycationic carrier particles. By using different hydrophilic monomers and hydrophilic monomers of different concentrations, the total net charge of the polycationic carrier particles can be adjusted, which in turn has an effect on the release kinetics and binding properties of the polycationic carrier particles.

[0133] In some embodiments, the hydrophilic monomer has Formula IX

[0134]

[0135] R6 is -O-(CH2) p -Y, -NJK, or -NJ2K + 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.

[0136] In some embodiments, the hydrophilic monomer is a monoacrylate monomer of formula X

[0137]

[0138] R8 is a C2-C4 straight chain alkyl group, 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.

[0139] In some embodiments, the acrylate monomer is cross-linked with one or more hydrophilic monomers at a molar percentage of 15 / 85 to 50 / 50, 20 / 80 to 40 / 60, or 25 / 75 to 35 / 65. In some embodiments, only one hydrophilic monomer is used, and the molar percentage of acrylate monomer to hydrophilic monomer is about 30 / 70. In one embodiment, two hydrophilic monomers are used, and the ratio of acrylate monomer to hydrophilic monomer 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 "±" applies to each value defined in the ratio or the total value of the ratio.

[0140] In some embodiments, the synthesis of charge transfer polycation carrier particles is carried out using a 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 hydrolyzable, unstable ester group. Similar acrylate crosslinkers that combine unstable esters with adjacent amino groups (e.g., according to Formula I-VIII) can also be used to prepare degradable particles and polymer materials. As shown in Formulas I-VIII above, examples of DXL derivatives include different spacer lengths containing tertiary or secondary amino groups, as well as triacrylate and tetraacrylate analogs. The loading percentage of DXL can be used to form particles with different sizes and different degradation rates. Copolymerization of DXL with DXL analogs can also be used to adjust the overall degradation rate of the particles.

[0141] The formation of controlled-size polymer particles can allow the formation of controlled-size polyplex particles (cationic particles plus payload). Heterogeneous polymerization systems for producing polymer particles include emulsion polymerization, dispersion polymerization, suspension polymerization, and precipitation polymerization. Typically, these heterogeneous polymerizations require the use of surfactants or steric stabilizers to form particles. These surface stabilizers can leave undesirable chemical forms on the particle surface groups, which may interfere with potential delivery applications.

[0142] Precipitation polymerization is an exemplary method for obtaining polycationic carrier particles at laboratory scale, but other methods for industrial scale and the like are also contemplated. Precipitation polymerization is a high yield method that can produce monodisperse, swellable, stabilizer-free particles suitable for biomedical applications. Precipitation polymerization is very suitable for making particles containing reactive monomers, especially water-sensitive monomers, and is very suitable for producing narrowly dispersed micron-sized particles without stabilizers or surfactants. In one embodiment, the yield is defined as the weight or molar ratio of the starting monomer and optional initiator to the monomer present in the formed polymer. In another embodiment, the yield is defined as the weight or molar ratio of the starting monomer and optional initiator to the monomer present in the particle. 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%. Polycationic carrier particles can be obtained by precipitation polymerization of acrylate monomers with one or more hydrophilic monomers under particle forming conditions, and then optionally hydrolyzed and / or functionalized. The acrylate monomers and / or the pendant acrylate groups of the hydrophilic monomers can be functionalized with small molecule thiols or amine compounds.

[0143] Precipitation polymerization begins with a homogeneous solution of monomers (acrylate monomers and one or more hydrophilic monomers) and initiators. Typical total monomer loadings are about 1 to about 20 weight % or about 2 to about 10 weight % of a solvent having a suitable solvency property for the formed polymer. When the total monomer loading is less than 1 weight %, the formation of particles becomes inefficient and limited. In one embodiment, one or more additional monomers (i.e., not acrylate monomers or hydrophilic monomers as defined herein) can be added to the monomer loading to produce a polymer suitable for a particular application.

[0144] The solvent for precipitation polymerization should be a poor enough solvent to cause the polymer to aggregate and form particles, but still a good enough solvent to swell the polymer chains on the surface of the particles, which prevents particle-particle aggregation during polymerization. In one embodiment, the Hildebrand solubility parameter of the solvent used is about 4 to about 5 MPa1 / 2 higher or lower than the Hildebrand solubility parameter of the formed polymer (i.e., higher or lower polarity). In addition, in some embodiments, the viscosity of the solvent is another factor considered when selecting a solvent. Low viscosity solvents are 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 above the polymerization temperature (typically 60-70°C for thermally initiated polymerization) and should not react substantially with the monomer or initiator. In the case of acrylate monomers, nucleophilic solvents such as water, alcohols or amines should be avoided. Particles can also be obtained from photoinitiated precipitation polymerization, which allows the use of lower boiling point solvents. 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, ether, dichloromethane, and pentane may be used.

[0145] Alternative methods for precipitation polymerization to obtain polycationic carrier particles include reverse emulsion polymerization, microemulsion polymerization, reverse suspension polymerization, and dispersion polymerization. Suspension polymer particles and reverse suspension polymer particles have homogeneous particle properties because they are formed in essentially micro bulk polymerization. Suspension polymerization and reverse suspension polymerization using mechanical dispersion of a liquid particle-forming phase (e.g., a monomer mixture) in a bulk continuous phase typically produce particles with a wide size distribution, given the statistical balance of droplet sharing and coalescence present in these processes. Reverse suspension polymerization of droplets of an aqueous solution of a hydrophilic monomer mechanically dispersed in an immiscible oily medium can be used to form spherical microparticles and microgels on a large scale, but with a wide size distribution. Similarly, dispersing an aqueous coacervate phase in a continuous aqueous phase, followed by crosslinking of the dispersed droplets into hydrogel beads, can be seen as an example of aqueous-aqueous suspension polymerization leading to spherical crosslinked hydrogel particles, but with a larger particle size distribution. Emulsion-type polymerization uses particle initiation in a continuous medium and can lead to large-scale production of narrowly dispersed nanoparticles. In both suspension and emulsion polymerizations, water is usually used as a solvent, which may not be suitable for reactive, hydrolytically unstable monomers. Dispersion polymerization begins with a solution of monomer, initiator, and colloidal stabilizer in a solvent that is hostile to polymer formation. The process takes advantage of the reduced solubility of the growing polymer chain and can be used to form monodisperse microparticles if large amounts of steric stabilizers are used to prevent aggregation of the formed particles.

[0146] The polycation carrier particles obtained can have a size of 200nm to 5μm. This size range is particularly suitable for 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 cellular uptake. The preferred range of the size of the particle is 0.5μm to 5μm, 1μm to 5μm, or 1μm to 3μm. The size can be defined as the diameter when the particle is spherical, or can be defined as the maximum distance between two relative points intercepted on a straight line passing through the center of the particle when the shape is irregular. The polycation carrier particles of the present disclosure generally have a spherical shape, wherein the surface is smooth or rough. 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.

[0147] When the polycationic carrier particle is internalized by the cell, it is exposed to specific aqueous or pH conditions that cause the polycationic carrier particle to degrade, for example, by chemical reactions of the reactive esters of the polycationic carrier particle. Reactive esters of acrylate monomers are susceptible to degradation by hydrolysis at pH 7 (but may also occur at other pHs). Generally, faster degradation will occur at higher pH (e.g., above 7), while slower degradation will occur at lower pH (e.g., below 7). The composition of the polycationic carrier particle can be altered by copolymerizing the above-mentioned different acrylates and hydrophilic monomers. Hydrophilic monomers can be used to alter the physical and chemical properties of the particles; hydrophilicity, hydrophobicity, charge, degradation rate.

[0148] When the polycationic carrier particles degrade, the crosslinks that hold the polymer chains together in the particles are broken and the polymer chains are released into the aqueous solution. As previously described, the polymer chains are composed of a portion of the now degraded acrylate monomers and small molecules. The polymer chains produced by particle degradation can be eliminated by the kidneys and cleared out of the subject's body. Degradation by hydrolysis can occur under physiological conditions (i.e., 37°C, pH 7) without the need for additional strong external stimuli. Degradation does not occur by free radicals. Therefore, degradation is considered biocompatible. When the DXL is an acrylate monomer, the polymer byproduct obtained will be an acrylic copolymer, a material commonly used in biomaterial applications (i.e., dental implants, hydrogels).

[0149] The material compounded with the polycationic carrier particles can be a vaccine, genetic material such as polynucleotides (mRNA, DNA, siRNA) or a therapeutic agent. The polycationic carrier particles can be used in aqueous environments where degradation may occur, such as delivering load materials within cells. The polycationic carrier particles can be used to bind proteins, such as ovalbumin, and other proteins such as growth factors. Negatively charged load materials can easily associate with positively charged particles, and are preferred (e.g., nucleic acids). The size of the particle, the charge of the particle, and the composition of the particle (monomer selection) can vary, thereby being optimized to a specific delivery route (e.g., intravenous or oral), and for delivery and dissociation of particles in a specific environment (e.g., pH range).

[0150] In some embodiments, the polycationic carrier particles are associated with or encapsulate the vaccine composition and can thus be used as a delivery platform for the antigen. Thus, the polycationic carrier particles can:

[0151] Serve as carriers for antigens, such as RNA (including m-RNA), DNA, proteins, viral capsid fragments, whole inactivated viral capsids, or active harmless viruses (such as adenoviruses that have been modified to express the desired antigenic protein);

[0152] Possess cationic groups that can electrostatically bind antigens to the recipient's immune system during storage and administration;

[0153] Possessing adjuvant properties to ensure recognition and processing by the host immune system, including where the adjuvant properties are based on cationic or polycationic groups, or on certain carbohydrate groups;

[0154] Having a composition in which the cationic or polycationic group can be cleaved by spontaneous or enzyme-mediated hydrolysis, thereby releasing the bound antigenic payload within a time frame that is favorable for eliciting a strong immune response in the recipient;

[0155] Having a cross-linker that undergoes slow spontaneous or enzyme-mediated hydrolysis, thereby ensuring that the particles are eventually cleared from the receptor by processes including renal clearance;

[0156] having a composition comprising cationic and polycationic groups and a non-stoichiometric amount of polyampholytes that can bind antigens to be stored at room temperature as defined up to 40°C and does not require cold chain logistics during storage and transportation; and

[0157] The polymer nanoparticles can be additionally loaded with silver nanoparticles to enhance cellular immune responses, either by co-precipitation during precipitation polymerization, or by reductive precipitation from silver salts as part of post-functionalization, or by adsorption of preformed silver nanoparticles onto the polymer nanoparticles.

[0158] Therefore, in some embodiments, a vaccine composition or therapeutic composition is provided, which comprises the polycationic carrier particles of the present disclosure. The polycationic carrier particles of the present disclosure can be administered to a subject in need without an additional 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 can be formulated as a part of an oral composition (e.g., oral capsule).

[0159] In some embodiments, the polycationic carrier particles are compounded with a load or payload that triggers an immune response and administered to a subject. The load of the polycationic carrier particles can be a nucleic acid, such as DNA and RNA. Therefore, in other embodiments, a method for delivering nucleic acids to cells by administering polycationic carrier particles of the present disclosure having a nucleic acid payload is provided. Therefore, polycationic carrier particles can be used for gene editing applications, such as gene knockout and gene editing in the case of clustered regularly interspaced short palindromic repeats (CRISPR), etc. In some embodiments, a method for gene therapy of a subject in need is provided, the method comprising administering a polycationic carrier particle of the present disclosure and a nucleic acid payload (e.g., in the form of a plasmid) to the subject.

[0160] The polycationic carrier particles of the present invention can also be used in antimicrobial materials, such as antimicrobial surfaces and coatings, and can also be used as flocculants in wastewater management.

[0161] Example

[0162] Material

[0163] 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, deuterium oxide (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's 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 TM The conjugate (OVA-RED), 1× phosphate buffered saline (PBS), and paraformaldehyde (16% (w / v) in methanol-free aqueous solution) were purchased from Thermo Fisher Scientific. The CellTiter 96 AQueous non-radioactive cell proliferation assay was purchased from Promega. 96-well glass bottom plates with high performance #1.5 cover glass were purchased from Cellvis. Aqueous hydrochloric acid (HCl) solution (35-37 wt %), sodium hydroxide (reagent grade), and sodium phosphate dibasic (≥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. Ethanol (95 vol %) was purchased from Commercial Alcohols.

[0164] Synthesis of Methyldiethanolamine Diacrylate (DXL)

[0165] In a round-bottom flask equipped with a magnetic stir bar, N-methyldiethanolamine (6 g, 50.4 mmol) and triethylamine (TEA) (12.714 g, 125.9 mmol) were dissolved in 400 mL of dichloromethane (DCM) and maintained at 0°C with 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, and the reaction mixture turned opaque and yellow after the addition. After the addition was complete, the reaction was stirred overnight and warmed to room temperature. The reaction mixture was washed 4 times with equal volumes of 5 wt% sodium bicarbonate and then 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 H nuclear magnetic resonance (NMR), the spectrum is shown in Figure 1 In, (CDCl3, 600MHz)d 6.37(2H,dd),6.10(2H,dd),5.80(2H,dd),4.23(4H,t),2.74(4H,t),2.35(3H,s).

[0166] Hydrolysis of DXL

[0167] A suspension of DXL at 0.75 wt% was prepared in 100 mM phosphate-buffered D2O and the pH was adjusted to pH 7.30 after dissolving the monomers in the buffer. The solution was transferred to a 5 mm NMR tube and recorded at different time intervals with a 500 MHz spectrometer from Bruker while keeping the sample at room temperature (about 22°C). 1 H NMR spectroscopy. The hydrolysis of DXL was measured by comparing the integrals of the N-methyl (-CH3) signal (δ 2.64 ppm), 2-((2-hydroxyethyl)(methyl)amino)ethyl acrylate (δ 2.93 ppm), and N-methyldiethanolamine (δ 2.97 ppm) of DXL in spectra recorded at different times and calculating the ratio of alcohol byproduct to diacrylate. Figure 2 ). Scheme 1 below shows the small molecule hydrolysis reaction scheme of DXL to form the corresponding alcohol byproduct and acrylic acid.

[0168] Solution 1. Ester hydrolysis of DXL forms 2-((2-hydroxyethyl)(methyl)amino)ethyl acrylate, N-methyldiethanolamine, and acrylic acid as byproducts

[0169]

[0170]

[0171] Synthesis of DXL cross-linked particles by precipitation polymerization

[0172] HEA-DXL (HD) particles: 2-Hydroxyethyl acrylate (HEA) and methyldiethanolamine diacrylate (DXL) were mixed to obtain a mixture containing 25-30 mol% DXL. The mixture was dissolved in a solvent mixture of methyl ethyl ketone (MEK) and heptane with a relative volume ratio of 70-85% MEK together with 1 mol% AIBN as a free radical initiator for thermal or photodecomposition at 5% monomer loading. A sample polymerization mixture targeting 70 mol% HEA and 30 mol% DXL in a 75 / 25 MEK / heptane solvent mixture at 1 g total monomer loading 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 and placed in a HB-1000 hybridization oven (UVP) set at 70°C with rotation mixing at 7 rpm for a total heating time of 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; Model 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 ambient temperature (22-25°C) for 20-24 hours.

[0173] After polymerization, the reaction mixture was transferred to a 50 mL centrifuge tube and diluted with an equal volume of 1,4-dioxane. The suspension was then centrifuged at 130 rcf for 15 minutes, after which 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 polymer, residual monomer, and initiator. In the case of 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 orange solids with an isolated yield of 5-10%.

[0174] HEA-DMAEA-DXL (HDD) particles: N,N-(Dimethylamino)ethyl acrylate (DMAEA), DXL and HEA were mixed at a relative monomer feed molar ratio of 1:1:1 D:D:H together with 1 mol% AIBN as a free radical initiator for thermal or photodecomposition, and then dissolved in a solvent mixture of methyl ethyl ketone (MEK) and heptane with a relative volume ratio of 70-85% MEK at 5% monomer loading. A sample polymerization mixture targeting a 1:1:1 molar ratio of D:D:H in a 75 / 25 MEK / heptane solvent mixture at 1 g total monomer loading 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 HD particles were isolated as described above. Dry HDD particles were obtained as orange solids with an isolated yield of 1-5%.

[0175] Optical Microscopy Program

[0176] HEA-DXL (HD) particles were imaged by bright field microscopy using a Nikon Eclipse LV100ND upright microscope, a Nikon Ti Eclipse inverted microscope, and a Nikon A1 confocal Ti Eclipse microscope. Images were analyzed using Nikon NIS-elements Advanced Research software version 5.11.01. Size measurements were manually determined using the three-point circle method (n=70).

[0177] Particle degradation solutions

[0178] DXL cross-linked particles were prepared at 0.1 wt% in PBS pH 7.4 and 0.1 M NaOH (pH about 11), respectively, to monitor particle degradation under physiological and accelerated conditions. The suspensions were kept at room temperature and imaged by bright field microscopy at different time points.

[0179] The particles were loaded with OVA (ovalbumin) (OVA-FITC (fluorescein isocyanate), OVA-RED)

[0180] The HD particles were sterilized by resuspending them in approximately 0.2 wt % 70% ethanol for 10 minutes. After sterilization, the particle solution was prepared and handled using aseptic techniques in a certified A2 Class 2 biosafety cabinet. The particle suspension in ethanol was then centrifuged at 130 rcf for 15 minutes, followed by removal of the supernatant and resuspended in sterile water. The washing procedure was repeated a total of 2 times to remove residual ethanol and form a sterile particle suspension in PBS with a final concentration of 0.2 wt %. Separately, a 0.2 wt % OVA-FITC solution was prepared in PBS and sterile filtered using a 0.22 mm syringe filter. To load the HD particles with OVA-FITC, 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 vortexing for 1 minute. The suspension was then centrifuged at 3200 rcf for 1 minute, the supernatant removed, and the pellet resuspended in 800 μL PBS. The washing procedure was repeated a total of 2 times, and the pellet was resuspended in 800 μL PBS to form HD particles loaded with OVA-FITC (HD-OVA-FITC) at a target concentration of 0.1% by weight. HD-OVA-FITC particles were then imaged by bright field and fluorescence confocal microscopy to visualize the binding of OVA-FITC to HD particles. HD particles were loaded with OVA-RED in the same manner as previously outlined for OVA-FITC.

[0181] Cellular uptake protocol

[0182] RAW 264.7 macrophages (ATCC) were cultured in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum and 1% v / v penicillin-streptomycin (100 units / mL) in T-75 tissue culture treated flasks and maintained at 37°C in a humidified atmosphere with 5% CO2. For cellular uptake assays, RAW 264.7 cells were seeded in glass-bottom 96-well plates (Cellvis) at a density of 8,000 cells / well (with a volume of 100 μL 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. 100 μL of a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin was added to the washed cells. As a control, cells were exposed to a) 100 μL serum-free DMEM, b) 100 μL of 10% v / v DMSO solution in serum-free DMIM, and c) 100 μL of 0.1% by weight OVA-FITC solution in serum-free DME (added to cells). Cells were incubated for 4 hours at 37°C in a humidified atmosphere with 5% CO2. After incubation, supernatant was removed from the cells, washed twice with about 250 mL of PBS, and then 100 μL of 4% v / v paraformaldehyde was added in sterile water. After 15 minutes, the paraformaldehyde solution was removed, and cells were washed twice with about 250 μL of PBS, and then 100 μL of PBS was added to the fixed cells. Cells were then imaged by bright field and confocal fluorescence microscopy.

[0183] MTS Assay Protocol (i.e. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, salt])

[0184] RAW 264.7 macrophages (ATCC) were cultured in DMEM supplemented with 10% v / v heat-inactivated fetal bovine serum and 1% v / v penicillin-streptomycin (100 units / mL) in T-75 tissue culture treated flasks and maintained at 37°C in a humidified atmosphere with 5% CO2. For cellular uptake assays, RAW 264.7 cells were seeded in tri-tissue culture (TC) treated plates at a density of 8,000 cells / well (with a volume of 100 μL 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. 100 μL of a 0.1 wt% HD-OVA-FITC particle suspension prepared in serum-free DMEM supplemented with 1% penicillin-streptomycin was added to the washed cells. As controls, 100 μL of serum-free DMEM supplemented with 1% penicillin-streptomycin, 100 μL of 10% v / v DMSO in serum-free DMIM supplemented with 1% penicillin-streptomycin, 100 μL of 0.1 wt% OVA-FITC in serum-free DMSM supplemented with 1% penicillin-streptomycin, and 100 μL of 0.1 wt% HD particle suspension in serum-free DMEM supplemented with 1.0% penicillin-streptomycin were added to the cells. The cells were incubated for 4 hours at 37°C in a humidified atmosphere with 5% CO2. 20 μL of MTS solution (Promega) was added to the wells and mixed. The plate was shaken for 10 seconds at a horizontal distance of 1 mm and tagged using a Biotek TM The absorbance was read at 490 nm using a 800TS absorbance reader.

[0185] Statistical analysis

[0186] Data were statistically analyzed using one-way analysis of variance (ANOVA) with Bonferroni post hoc analysis. The significance level was set at p < 0.05.

[0187] Hydrolysis of DXL

[0188] DXL was hydrolyzed alone to model the hydrolysis and degradation of DXL cross-linked particles. Degradation of DXL can occur at two reactive ester sites per DXL unit, and it should be noted that hydrolysis of only one of the ester groups is sufficient to degrade the crosslink. Furthermore, hydrolysis of the second ester group of the same unit will release N-methyldiethanolamine as a small molecule byproduct, while the acrylic acid unit will remain on the polymer.

[0189] pass 1H NMR spectroscopy was used to monitor the hydrolysis of DXL in phosphate-buffered D2O at pH 7.3 maintained at room temperature (22°C). The results showed that after 1 day at room temperature (22°C), about 31% of DXL was hydrolyzed ( Figure 3 A small portion of the monoacrylate product had been further hydrolyzed to give N-methyldiethanolamine (MDEA), resulting in a product consisting of 27.2% monoacrylate and 3.4% MDEA. The hydrolysis of one of the ester groups of DXL showed linear pseudo-first order kinetics with a rate constant (k) of 0.0151 h -1 And the half-life is about 1.91 days ( Figure 4 ).

[0190] Results indicate that at pH 7, DXL hydrolyzes approximately 1.8 times faster than poly[N,N-dimethylaminoethyl acrylate] (PDMAEA), which has a half-life of approximately 3.5 days. Hydrolysis of polymerized DXL within the particles is expected to occur at a similar rate, with the presence of comonomers and polarity within the particles potentially influencing the rate. Adjacent functional groups incorporated into the DXL structure or comonomers may also be used to modulate the hydrolysis and degradation rates of the corresponding particles.

[0191] Precipitation polymerization of DXL cross-linked particles

[0192] DXL is used as a diacrylate crosslinker to form degradable cationic particles in aqueous media. DXL consists of beta-amino ester bonds, which have been shown to undergo rapid ester hydrolysis under mild physiological conditions. N,N-(dimethylaminoethyl)acrylate (DMAEA) is an example of a monomer with such a reactive ester that has been studied in the field of degradable polymeric materials. It is hypothesized that DXL crosslinked particles can be used as a delivery vehicle for therapeutic payloads such as DNA / RNA and proteins due to its cationic charge with tertiary ammonium groups. Thus, DXL loaded particles can promote cellular uptake through the net cationic charge and then degrade intracellularly through hydrolysis of the labile ester bonds, thereby releasing the complexed payload. Degradation reduces the cationic charge on the polymer chain, and the soluble polymer can be cleared from the body by renal filtration.

[0193] Precipitation polymerization was chosen to prepare DXL cross-linked particles because it does not require the use of surfactants or steric stabilizers compared to other methods such as emulsion polymerization. DXL was polymerized with HEA as a neutral polar comonomer in a marginal solvent system consisting of MEK as a polar good solvent and heptane as a non-polar poor solvent. When HEA-DXL polymer chains were initially formed in the marginal solvent, aggregation of polymer chains formed particle cores, which captured subsequently formed polymer chains to grow polymer particles. A HEA / DXL molar feed ratio of 70 / 30 was chosen due to the limited solubility of DXL in a 75 / 25 MEK / heptane solvent system that was found to be optimal for particle formation. This formulation was prepared by photo-initiated and thermally-initiated free radical polymerization. The size of the particles was 2.28±0.35 μm (n=70) for the photo-initiated polymerization and 3.60±1.45 μm (n=70) for the thermally-initiated polymerization (see Figures 2 and 3, respectively). Figure 5 and Figure 6 ). It should be noted that the samples prepared by thermal polymerization were sonicated to separate the particles from larger aggregates.

[0194] Compared with those particles formed by thermal polymerization, particles formed by photopolymerization have smaller diameters and narrower diameter dispersion. Particles formed by photopolymerization are also more stable in space.

[0195] Since DXL showed limited solubility in MEK / heptane solvent mixture compositions 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 particle system (referred to herein as HDD) was tried with a 85 / 15 MEK / heptane solvent mixture at a 1 / 1 / 1 molar feed ratio and polymerized by thermal and photopolymerization. The HDD particles obtained by thermal and photopolymerization are shown in Figures 1 and 2, respectively. Figure 7 and Figure 8 middle.

[0196] HDD particles prepared by thermal polymerization produced a mixture of particles and particle aggregates (n=70) with a diameter of 1.85±0.47 μm. HDD particles of the same composition prepared by photopolymerization produced submicron particles that could not be analyzed by bright field microscopy.

[0197] Similar to HD particles, the size of HDD particles with the same composition was smaller when prepared by thermal polymerization compared to photopolymerization. This difference was again attributed to the different polymerization rates and extents as well as the effect of polymerization temperature on the solvency.

[0198] Due to the stability of the obtained particle suspension and the relatively narrow size dispersity of the sample, HD particles prepared by photopolymerization were selected for further proof-of-concept experiments. After purification, the HD particles were resuspended in PBS, pH 7.4, which produced a mixture of particles and particle aggregates with a diameter of 2.21 ± 0.44 μm (n = 70) ( Fig. 9 ).

[0199] Loading HD particles with OVA (OVA-FITC)

[0200] Due to the net cationic charge of the tertiary ammonium groups of DXL in aqueous media, HD particles can bind to proteins as well as anionic materials such as DNA / RNA. Due to the labile ester bond of HD particles to DXL, the particles can degrade and release the bound payload. To demonstrate the ability of the particles to bind proteins for potential drug delivery applications, HD particles were exposed to FITC-labeled ovalbumin (OVA-FITC) or Texas Red-labeled ovalbumin (OVA-CED).

[0201] Confocal fluorescence microscopy of unloaded HD particles showed that autofluorescence was detected from the particles with maximum intensity in the FITC channel. The fluorescence of the particles is likely due to DXL, as polymers containing tertiary amines have been shown to exhibit fluorescent properties. Under the same acquisition settings, the fluorescence of HD particles in the TRITC channel was weaker ( Figures 10A-10D ).

[0202] Therefore, to minimize interference from HD autofluorescence, OVA-RED was used for binding studies. HD particles loaded with OVA-RED at a 1:1 weight ratio (HD-OVA-RED) were imaged by bright field and confocal fluorescence microscopy and the results are shown in Figures 11A-11D When HD particles without OVA-RED were imaged under the same settings, the signal in the TRITC channel was much weaker ( Figures 12A-12B ), indicating that HD particles are indeed able to bind to proteins such as OVA. Note that after HD particles were bound to OVA-RED, some aggregates of insoluble OVA-RED that were not bound to HD particles were observed ( Fig. 12A ). This may be due to the reduced solubility of the Texas Red moiety in aqueous media.

[0203] Cellular uptake

[0204] HD particles loaded with OVA-FITC were exposed to RAW 264.7 macrophages as an in vitro model for vaccine delivery. It was hypothesized that HD-OVA particles would be taken up by immune cells and then degraded intracellularly by DXL hydrolysis to release the loaded antigen. Other cationic delivery vehicles based on non-charge transfer polycations have shown promise in binding antigens but often pose challenges in releasing antigens. Non-charge transfer polycations also pose a potential risk of damaging cells and cellular components after delivery due to the toxicity of such polymers.

[0205] HD-OVA-FITC particles were incubated with RAW 264.7 cells for 4 hours, and then the excess particles in the solution were removed by washing twice with PBS. The cells were then imaged by bright field microscopy ( Fig.13A -C). Fig.13A It is shown that the morphology of cells treated with HD-OVA-FITC particles is similar to that of untreated cells incubated in DMEM ( Fig. 13C ). Fig.13A It is also shown that a portion of the HD-OVA-FITC particles appear to have aggregated to form larger clusters. As a negative control, cells were incubated with 10% v / v DMSO in DMEM and showed round cell morphology and signs of apoptosis ( Fig. 13B ).

[0206] Because autofluorescence from HD particles and cells may contribute to the signal in the FITC channel, images of individual cells were collected ( Figures 14A-14D ) and images of cells treated with unloaded HD particles ( Figures 15A-15D ). In both cases, no obvious fluorescence was observed from the cells, except for some particle aggregates on the surface of individual cells in FIG15 .

[0207] Next, cells were imaged by confocal fluorescence microscopy to examine the internalization of HD-OVA-FITC particles. Figures 16A-16D Images of cells treated with HD-OVA-FITC and then stained with NucBlue nuclei are shown in Figure 1. Fluorescence in the FITC channel was observed from the cells (either as diffuse areas or punctate areas in the cells), indicating that OVA-FITC had been internalized. Colocalization of HD-OVA-FITC particles around the cell nucleus was observed by confocal microscopy of the merged FITC and DAPI channels ( Fig.17 ). This indicates that OVA-FITC has successfully entered macrophages. These results suggest that macrophages will preferentially take up cationic particles because they interact with the negatively charged cell membrane.

[0208] The cytotoxicity of HD-OVA-FITC particles on RAW 264.7 cells was evaluated by using an MTS proliferation assay. As controls, cells were incubated with serum-free DMEM, 0.1 wt% HD particle suspension prepared in serum-free DMIM, and 10% v / v DMSO in serum-free DMEM (all supplemented with 1% penicillin-streptomycin). After 4 h of incubation, the average viability of RAW 264.7 cells was 106 ± 31%, 100 ± 22%, 117 ± 23%, 64 ± 12%, and 10% v / v DMSO, respectively. Fig.18 ).

[0209] The decreased survival rate of RAW 264.7 cells in 10% v / v DMSO was statistically significant (p<0.05) compared to any sample containing particles (0.1 wt% HD-OVA-FITC, 0.1 wt% HD), while the survival rate of RAW 264.7 cells in serum-free DMEM was not statistically significant compared to any sample containing particles (p>0.05). The results indicate that HD particles loaded with and without OVA have no significant cytotoxicity at 0.1 wt%, as the survival rate of the managed cells is comparable to that of the control cells in serum-free medium.

Claims

1. A polycationic carrier particle obtained by polymerizing an acrylate monomer with one or more hydrophilic monomers to form a crosslink, wherein the polycationic carrier particle has a net positive charge, and wherein the acrylate monomer is selected from Formula I or Formula V and wherein R and R" are each independently hydrogen, C1-C 14 a linear or branched alkyl group, a C3-C8 cycloalkyl group or a 5- to 10-membered aryl or heteroaryl ring, optionally terminated by a polymerizable group, preferably an acrylate group, capable of crosslinking with one or more hydrophilic monomers, R' is C2-C 14 a linear or branched alkyl group, a C3-C8 cycloalkyl group or a 5- to 10-membered aryl or heteroaryl ring, R' being terminated by at least one polymerizable group capable of crosslinking with one or more hydrophilic monomers, preferably an acrylate group, The alkyl, cycloalkyl, aryl and heteroaryl groups can be optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and R2 is C2 or C3, and wherein C3 is linear or cyclic.

2. The polycationic 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 polycationic carrier particle according to claim 1 or 2, wherein the acrylate monomer of formula I is a multi-acrylate monomer of formula II Where R1 is hydrogen or C1-C 14 linear, branched or cyclic alkyl, optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms and optionally terminated by an acrylate group, R2 is C2 or C3, and wherein C3 is linear or cyclic, R3 is C 2- C 14 Straight or branched chain alkyl, which is optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

4. The polycation carrier particle according to claim 3, wherein the multi-acrylate monomer is of formula III Wherein R1 and R2 are as defined in claim 2, and R4 is -CH2-CH2-O- or -CH2-CH2-CH2-O-, the terminal CH2 group of R4 is connected to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3 or 4.

5. The polycationic carrier particle according to claim 3, wherein the multi-acrylate monomer is of formula IV wherein R1 and R2 are as defined in claim 2, R1′ has the same definition as R1, R2′ is C2-C3 wherein C3 is linear or cyclic, and m is an integer selected from 0, 1, 2, 3 or 4.

6. The polycationic carrier particle of any one of claims 1 to 5, wherein the acrylate monomer of formula V is a multiacrylate monomer of formula VI Wherein R1 and R5 are independently hydrogen or C1-C 14 a linear, branched or cyclic alkyl group, optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms, and optionally terminated by an acrylate group, R2 is C2 or C3, wherein C3 is linear or cyclic, R3 is C2-C 14 Straight or branched chain alkyl, optionally substituted and optionally interrupted by one or more oxygen, sulfur or nitrogen atoms.

7. The polycationic carrier particle according to claim 6, wherein the multi-acrylate monomer is of formula VII Wherein R1 and R5 are as defined in claim 6, and R4 is -CH2-CH2-O- or -CH2-CH2-CH2-O-, the terminal CH2 group of R4 is connected to the oxide of the terminal acrylate group, and n is an integer selected from 1, 2, 3 or 4.

8. The polycationic carrier particle of claim 5, wherein one or more nitrogen atoms are protonated.

9. The polycationic carrier particle according to claim 8, wherein the multiacrylate monomer has formula VIII wherein R1 and R5 are as defined in claim 6, R2 is C2 or C3, and wherein C3 is linear or cyclic, m is an integer selected from 0, 1, 2, 3 or 4, and R1', R2' and R5' independently share the definitions of R1, R2 and R5.

10. The polycationic carrier particle according to claim 1, wherein the acrylate monomer is selected from:

11. The polycationic carrier particle according to any one of claims 1 to 3, wherein the multiacrylate monomer is methyldiethanolamine diacrylate (DXL).

12. The polycationic carrier particle according to any one of claims 1 to 11, wherein the hydrophilic monomer is of formula IX Where R6 is -O-(CH2) p -Y, -NJK, or -NJ2K + , p is an integer selected from 2, 3 or 4, and Y is defined as OH, COOH, NJ2, NJ3 + or ONJ2, and each J is independently defined as H, methyl or ethyl, and K is optionally branched and optionally substituted ethyl, propyl or butyl, and R7 is hydrogen or methyl.

13. The polycationic carrier particle according to any one of claims 1 to 12, wherein the hydrophilic monomer is of formula X wherein R8 is a C2-C4 straight chain alkyl group, and X is selected from hydroxyl, carboxyl, tertiary amine, quaternary ammonium or amide, and the amine is optionally substituted with methyl or ethyl.

14. The polycationic carrier particle according to any one of claims 1 to 13, wherein the one or more hydrophilic monomers are 2-hydroxyethyl acrylate (HEA) and / or N,N-(dimethylamino)ethyl acrylate (DMAEA). 15 . The polycationic carrier particle according to claim 1 , wherein the polycationic carrier particle has a size of 200 nm to 5 μm.

16. The polycationic carrier particle according to any one of claims 1 to 15, wherein the molar percentage of the acrylate monomer to the one or more hydrophilic monomers is 15 / 85 to 50 / 50.

17. The polycationic carrier particle according to any one of claims 1 to 16, further comprising a support material complexed with the polycationic carrier particle.

18. A complex comprising polycationic carrier particles complexed with load molecules, the polycationic carrier particles being suitable for releasing the load molecules within cells and being degradable under physiological conditions, and wherein the polycationic carrier particles have charge transfer 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 preparing a polycationic carrier particle as defined in any one of claims 1 to 17, the method comprising: providing an acrylate monomer as defined in any one of claims 1 to 11 and one or more hydrophilic monomers as defined in any one of claims 1 to 14, The acrylate monomer is cross-linked with the one or more hydrophilic monomers.

20. The method of claim 19, wherein the cross-linking step comprises performing one of precipitation polymerization, mini-emulsion polymerization, dispersion polymerization, inverse suspension polymerization, or inverse 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 of the acrylate monomer to the hydrophilic monomer is 30±15 / 70±15.

22. The method according to any one of claims 19 to 21, wherein two hydrophilic monomers are provided, and the molar percentage of the acrylate monomer to the two hydrophilic monomers is 33±10 / 33±10 / 33±10.

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

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

25. The method of any one of claims 19 to 24, further comprising providing an agent to be encapsulated and crosslinking the acrylate monomer with the one or more hydrophilic monomers in a medium containing the agent.

26. A method for producing a composite of a load material and a polycationic carrier particle, comprising: Producing the polycationic carrier particles by the method according to any one of claims 19 to 24; as well as The composite is obtained by exposing the polycation carrier particles to a solution containing a support material, thereby complexing the negatively charged support material to the positively charged polycation carrier particles.

27. A method of delivering a vaccine to a subject in need thereof, the method comprising administering to the subject the polycationic carrier particle of any one of claims 1 to 17, wherein the polycationic carrier particle is complexed with a vaccine agent or encapsulates a vaccine composition.

28. A method of inducing an immune response in vivo, comprising administering to a subject in need thereof the polycationic carrier particle of any one of claims 1 to 17, said polycationic carrier particle being complexed with an antigen or a nucleic acid encoding an antigen or encapsulating a vaccine composition.

29. A method of delivering nucleic acids for in vivo protein expression, comprising administering to a subject the polycationic carrier particle of any one of claims 1 to 17, wherein the polycationic carrier particle is complexed with a nucleic acid encoding a protein.

30. A vaccine composition comprising the polycationic carrier particle of any one of claims 1 to 17 complexed with an antigen or a nucleic acid encoding the antigen and a pharmaceutically acceptable carrier or adjuvant.

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

32. The complex of claim 18, wherein the load molecule is selected from the group consisting of nucleic acids, peptides, and proteins.

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