Uses of reactive microparticles and functional hydrogel particles for preparation

Abstract

A method for producing hydrogel microparticles with a spherical shape and a narrow or monodisperse size distribution is provided. At least one temporary crosslinker, such as those represented by formulas (I), (IIa), (IIb), (IIc), (IId), and (IIc) are combined with at least one permanent crosslinker containing two or more vinyl groups, such as divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), N,N'-methylenebisacrylamide (MBA), oligo / polyethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 1,6-hexanediol dimethacrylate, in an organic solvent with a polarity suitable for precipitation polymerization. The precipitation polymerization occurs without the addition of surfactants and / or stabilizers, and / or the formed microparticles contain less than 1% surfactants and / or stabilizers. These microparticles can be further functionalized by functionalizing the monomers of the temporary crosslinker to provide amine and carboxylic acid units. The functionalized microparticles are used for cryopreserving cells or as vaccine delivery platforms. [Formula 1] JPEG2023552661000044.jpg100108

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 122,322, filed on 7 December 2020, the contents of which are incorporated herein by reference.

[0002] The present invention aims to form and use hydrogel microparticles having charged groups. More specifically, the present invention relates to the formation of reactive crosslinked microparticles that can be converted into functional hydrogel crosslinked microparticles. [Background technology]

[0003] Because hydrogels are recognized as being able to mimic several properties of natural tissues, they have long been recognized as useful materials for cell and tissue interfaces, and can therefore be considered synthetic extracellular matrix (ECM) materials. Examples range from hydroxyethyl methacrylate (HEMA)-based synthetic cross-linked hydrogels used as contact lenses, to Matrigel®, a commercially available ECM derived from mouse cancer cell lines that enables stem cell culture without inducing differentiation, and many forms of polyethylene glycol-based hydrogels.

[0004] In addition to bulk hydrogels, many techniques and applications have been described for hydrogels formed as irregular or spherical particles with narrow or wide size distributions in the nanometer and micrometer size ranges. Such microgel particles have attracted considerable interest for many potential applications.

[0005] Microgels have been used as cryoprotective materials for the cryopreservation of mammalian cells. For example, betaine-functionalized crosslinked hydrogels are mechanically broken down into irregularly shaped microparticles that exhibit cryoprotective properties. Cell adhesion within a matrix constructed with reverse suspension microgels having a broad particle size distribution has been demonstrated. 1

[0006] Several polymer particles in the micron and submicron ranges have been found to be useful as virus-like particles (VLPs) that enhance antigen uptake by the host immune system.

[0007] Both the bulk and microparticle forms of hydrogels need to possess desirable mechanical properties and be able to be fine-tuned to exhibit appropriate chemical and biological groups for biomedical applications. One method for fine-tuning hydrogel properties is through the use of reactive precursor particles that can be functionalized as desired before hydrogel formation. Polymer particles are typically produced by suspension, emulsification, dispersion, or precipitation polymerization.

[0008] Polymer particles can be obtained by splitting larger polymer solids or gels into smaller pieces, or by controlling phase separation of pre-formed polymers from solutions. However, in most cases, spherical polymer particles are formed using particle-forming polymerization methods such as suspension, reverse (water in oil) suspension, emulsification, reverse emulsification, dispersion, or precipitation polymerization.

[0009] Suspension and reverse suspension polymer particles have homogeneous particle properties because they are essentially formed by minibulk polymerization. However, stabilizers remaining on their surfaces may affect their interactions with cells and tissues. Similarly, suspension and reverse suspension polymerization carried out using the mechanical dispersion of a liquid particle-forming phase (e.g., monomer mixture) in a bulk continuous phase usually produces particles with a wide size distribution, given the statistical balance of droplet sharing and coalescence observed in these methods.

[0010] Spherical microparticles and microgels can be formed on a large scale using reverse suspension polymerization of droplets of aqueous solutions of hydrophilic monomers mechanically dispersed in an immiscible oil-like medium, but they have a wide size distribution.

[0011] Similarly, after dispersing an aqueous coacervate phase in a continuous aqueous phase and subsequently crosslinking the dispersed droplets into hydrogel beads, it can be seen as an example of aqueous-aqueous suspension polymerization that produces spherical crosslinked hydrogel particles, but still has a large particle size distribution. 2,3

[0012] Emulsion polymerization can use initiators of particles in a continuous medium and can result in the large-scale production of narrow-dispersed nanoparticles. In both suspension polymerization and emulsion polymerization, water is typically used as a solvent and may not react with reactive monomers that are unstable to hydrolysis.

[0013] Dispersion polymerization starts with a solution of monomers, initiators, and colloidal stabilizers in a poor solvent for the polymer being formed. This method utilizes the decreasing solubility of growing polymer chains and can be used to form monodispersed microparticles when a large amount of steric stabilizers are used to prevent aggregation of the particles being formed.

[0014] Microfluidic particle formation is a version of suspension polymerization that involves one-by-one formation of micrometer-sized droplets of a monomer or polymer solution suspended in a continuous medium, followed by subsequent rapid curing or crosslinking. Such methods have been used to prepare narrow-dispersed and monodispersed hydrogel particles for use as supports in the culture of β cells. 4 The ability of 20-micrometer hydrogel beads has been shown to support cell adhesion via the RGD cell adhesion motif and increase cell viability due to several factors including better oxygen diffusion. The disadvantages of this method are that, considering the formation principle per particle, there is a limit to throughput, stabilizers are required, and particles with radial crosslinking and other compositional gradients cannot be produced.

[0015] Stabilizers and surfactants used in dispersion, suspension, inverse suspension, and emulsion polymerization, including microfluidic deformation, can be incorporated into particles, and their presence, particularly at the particle surface, may affect the subsequent use of the particles.

[0016] Precipitation polymerization is a variant of dispersion polymerization without the addition of a colloidal stabilizer. As a result, the growing polymer chains aggregate in an uncontrolled manner, resulting in irregularly shaped particles with a wide size distribution.

[0017] Controlled precipitation polymerization is typically a variant of precipitation polymerization that uses a significant amount of crosslinking agent, and the polarity and hydrogen bonding ability of the solvent or solvents are adjusted relative to the polymer being formed such that the growing polymer chains aggregate to form nuclei and are colloidal-stabilized by the solvation surface layer of the chains that have just been absorbed. These particle nuclei then grow in parallel by absorbing more polymer and monomer to form a final population of microparticles in the diameter range of 0.3 to 20 micrometers.

[0018] The total monomer dosage in precipitation polymerization tends to be limited to 2 to 20, more usually 2 to 10 weight percent of the total monomer in the solvent. Higher monomer dosages typically cause the formation of particle agglomerates, and lower monomer dosages result in low yields due to inefficient particle nucleation and growth.

[0019] One skilled in the art can adjust the solvent polarity to affect the number of polymer nuclei present at the point where this colloidal stabilization occurs, thereby controlling the final particle size. One of the monomers used in precipitation polymerization must be a crosslinking agent. The presence of such a crosslinking agent has significant implications for capturing oligomers in the nuclei and subsequently on the growing particles. The presence of the crosslinking agent also helps to maintain particle integrity during growth.

[0020] Particle yields in precipitation polymerization are highest with high crosslinking agent content compared to other monomers. However, particles produced with high crosslinking agent content are typically not deformable enough for the target application. When low crosslinking agent content is used, the resulting yield is often very low (e.g., <10%). 5 This method is suitable only on a small scale (i.e., experimental or laboratory scale). If the yield is too low due to the low content of the crosslinking agent, a commercially and economically viable method cannot be obtained.

[0021] Therefore, lightly crosslinked swellable particles are typically formed in low yields using this method. This is because the low crosslink density required for swellable particles is incompatible with the need for a large amount of crosslinking agent during polymerization to form particles in high yield. High yields are necessary to derive any usefulness on an industrial scale.

[0022] Functional groups are generally introduced by the selection of appropriate comonomers, rather than by functionalization after the formation of pre-formed particles. Therefore, means of functionalizing microgel particles to meet specific needs are not always available. [Overview of the project]

[0023] In one embodiment, a method for generating microparticles is provided, comprising the steps of: combining at least one temporary crosslinking agent and at least one permanent crosslinking agent in an organic solvent having polarity suitable for controlled precipitation polymerization; and causing precipitation polymerization to occur, thereby forming microparticles having a polymer containing monomers of the temporary and permanent crosslinking agents.

[0024] In one embodiment, the total monomer content before precipitation polymerization is calculated as the combined content of at least one temporary crosslinking agent, at least one permanent crosslinking agent, and any other monomers, and has a value of 1 to 20% by weight.

[0025] In one embodiment, the total amount of crosslinking agent before precipitation polymerization is the combined amount of temporary and permanent crosslinking agents, and is greater than 10 mol%, with the ratio of temporary crosslinking agent to permanent crosslinking agent being 50:50 to 99:1 mol%.

[0026] In one embodiment, the solvent is 4-5 MPa higher than the polymer value. 1 / 2 High or low. In one embodiment, the solvent is selected from the group consisting of acetonitrile, methyl ethyl ketone, heptane, and combinations thereof. In another embodiment, the solvent is selected from the group consisting of acetonitrile, methyl ethyl ketone, heptane, and combinations of methyl ethyl ketone and heptane.

[0027] In one embodiment, the temporary crosslinking agent is formula (I) or (IIa)~(IIf)

[0028] [ka] [In the formula, R1 and R2 are independently selected from H, a C1-C4 linear or branched carbon chain, benzyl, phenyl, or OJ, and J is defined as a C1-C4 linear or branched carbon chain].

[0029] [ka] [In the formula, n is an integer between 1 and 3].

[0030] [ka] [In the formula, R3 is independently H or methyl] is a crosslinking agent.

[0031] In one embodiment, the temporary crosslinking agent is methacrylic anhydride or acrylic anhydride.

[0032] In one embodiment, the permanent crosslinking agent has two or more vinyl groups. In one embodiment, the permanent crosslinking agent is selected from the group consisting of divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), and N,N'-methylenebisacrylamide (MBA).

[0033] In one embodiment, the permanent crosslinking agent is present in an amount of 1 to 30 mol% of the total monomer content.

[0034] In one embodiment, the yield of microparticles is at least 30%, preferably at least 50%.

[0035] In one embodiment, precipitation polymerization is carried out in the absence of surfactants and / or stabilizers. In one embodiment, the method is carried out without the addition of surfactants and / or stabilizers.

[0036] In one embodiment, the microparticles have an outer surface containing less than 3% surfactant and / or stabilizer.

[0037] In one embodiment, the method further includes the step of functionalizing monomer units within particles derived from a temporary crosslinking agent.

[0038] In one embodiment, the functionalization step includes functionalizing the amine and carboxylic acid units in a ratio of 3:1 to 1:3. In one embodiment, the ratio is 2:1 to 1:2.

[0039] In another embodiment, microparticles comprising at least one polymer, where at least one polymer is Formulas (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), (IIIn), and / or (IIIo)

[0040] [ka] [In the formula, R1 and R2 are independently selected from H, a C1-C4 linear or branched carbon chain, benzyl, phenyl, or -OJ, J is defined as a C1-C4 linear or branched carbon chain, and the dashed line represents the remainder of the polymer backbone.]

[0041] [ka]

[0042] [ka] [In the formula, n is an integer between 1 and 3].

[0043] [ka]

[0044] [ka] The present invention provides microparticles comprising a temporary crosslinking monomer and a permanent crosslinking monomer [wherein R3 is independently H or methyl], wherein the microparticles are narrowly dispersed or monodisperse and have a size distribution with a coefficient of variation of less than 0.3.

[0045] In one embodiment, the hydrogel microparticles do not contain any detectable surfactants or stabilizers.

[0046] In some embodiments, the hydrogel microparticles contain less than 3%, preferably less than 1%, of surfactants and / or stabilizers.

[0047] In some embodiments, the hydrogel microparticles have a surface and a core, and less than 3%, preferably less than 1%, of the surface area is a surfactant and / or stabilizer.

[0048] In one embodiment, the hydrogel microparticles have a wet-to-dry swelling ratio of 5:1 to 50:1.

[0049] In one embodiment, the hydrogel microparticles have a total crosslinking agent content of 1 to 20 mol%, preferably 5 to 15 mol%, relative to the total monomer content.

[0050] In one embodiment, the hydrogel microparticles have a deformability of 1 kPa to 500 kPa, preferably 10 to 100 kPa.

[0051] In one embodiment, the hydrogel microparticles have a spherical shape when they swell in an aqueous medium.

[0052] In one embodiment, the hydrogel microparticles have a diameter of 0.5 to 20 micrometers.

[0053] In one embodiment, the hydrogel microparticles have a diameter of 1 to 10 micrometers.

[0054] In one embodiment, the permanent crosslinking monomer is a monomer of divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), oligo / polyethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, 1,6-hexanediol dimethacrylate, N,N'-methylenebisacrylamide (MBA), oligo / polyethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 1,6-hexanediol dimethacrylate.

[0055] In one embodiment, the temporary crosslinking agent monomer is a monomer of methacrylic anhydride and / or acrylic anhydride.

[0056] In yet another embodiment, microparticles produced by the method described herein are provided.

[0057] In an additional embodiment, a method for cryopreserving cells is provided, comprising the steps of preparing microparticles as described herein, functionalizing the microparticles, contacting cells with the microparticles, and freezing the cells.

[0058] In a further embodiment, a method for generating a vaccine delivery platform is provided, comprising the steps of: preparing microparticles as described herein; functionalizing the microparticles to act as carriers for an antigen; and linking an antigen to the carrier.

[0059] In another embodiment, a method for generating encapsulated cells is provided, comprising the steps of preparing microparticles as described herein, functionalizing the microparticles, combining the functionalized microparticles with cells and a capsule-forming material, and gelling the capsule-forming material so that the particles and cells are captured within the capsule. In one embodiment, the capsule-forming material is an alginate.

[0060] In yet another embodiment, a cryopreservation agent for cells is provided, comprising a monodisperse composition of biocompatible amphoteric polymer electrolyte hydrogel microparticles, wherein the hydrogel microparticles have a deformability of 100 Pa to 100 kPa, preferably 1 to 10 kPa, are substantially free of surfactants or stabilizers, and have a wet-to-dry swelling ratio of 5:1 to 50:1.

[0061] In an additional embodiment, a method for cryopreserving cells is provided, comprising the steps of combining a monodisperse composition of biocompatible amphoteric polyelectrolyte hydrogel microparticles described herein with cells in an aqueous suspension at a microparticle-to-cell volume ratio of 5000:1 to 10:1, preferably 1000:1 to 100:1; and freezing the suspension of microparticles and cells.

[0062] In one embodiment, freezing is carried out at a rate of 1°C / min down to -80°C, after which the cryotube is optionally moved to a storage container maintained at the liquid nitrogen evaporation temperature.

[0063] In another embodiment, the cell suspension is rapidly frozen by immersion in an environment maintained at -70 to -80°C.

[0064] In yet another embodiment, the cell suspension is frozen by immersion in an environment maintained at the evaporation temperature of liquid nitrogen, which is -195.6°C at atmospheric pressure.

[0065] In another embodiment, the cells are stem cells. In yet another embodiment, the cells are primary cells.

[0066] In another embodiment, the hydrogel microparticles are concentrated at a concentration of 1-25 wt / v%.

[0067] In another embodiment, the cells are clusters of cells, each containing 10 to 5,000 cells, preferably 100 to 2,000 cells, and are also known as organoids.

[0068] In a further embodiment, the present invention provides a vaccine delivery vehicle comprising a monodisperse composition of biocompatible hydrogel microparticles, wherein the hydrogel microparticles are cationic or amphoteric polyelectrolytes having an excess cationic charge, substantially free of added surfactants or stabilizers, having a wet-to-dry swelling ratio of 5:1 to 50:1, and having an average particle diameter of 0.1 to 10 microns.

[0069] In one embodiment, the microparticles can be decomposed under physiological conditions over a period of 30 minutes to 10 days, preferably 2 to 48 hours.

[0070] In one embodiment, a method for producing a vaccine is provided, comprising the step of combining a vaccine delivery vehicle described herein with an antigen.

[0071] In another embodiment, a granular extracellular matrix comprising a monodisperse composition of biocompatible hydrogel microparticles is provided, wherein the hydrogel microparticles have a deformability of 100 to 100 kPa, preferably 1 to 10 kPa, a surface substantially free of surfactants or stabilizers, and a wet-to-dry swelling ratio of 5:1 to 50:1. In one embodiment, the microparticles are modified with cell adhesion molecules.

[0072] In yet another embodiment, the present invention provides a method comprising the steps of adding a monodisperse composition of biocompatible hydrogel microparticles described herein to a suspension of mammalian cells in a cell-to-microparticle ratio of 1:100 to 1:1 in a gel-forming agent, and gelling the suspension.

[0073] In another embodiment, a cell culture method is provided, comprising the steps of preparing a granular extracellular matrix as described herein and growing a cell culture on the granular extracellular matrix.

[0074] In yet another embodiment, the present invention provides biomimetic beads comprising biocompatible hydrogel microparticles and biomimetic functional groups, wherein the hydrogel microparticles have a deformability of 100 to 100 kPa, preferably 1 to 10 kPa, are substantially free of added surfactants or stabilizers, and have a wet-to-dry swelling ratio of 5:1 to 50:1.

[0075] In yet another embodiment, a cell culture method is provided which includes the steps of providing a cell culture with biomimetic beads as described herein and growing the cell culture. [Brief explanation of the drawing]

[0076] [Figure 1]This reaction scheme demonstrates the polymerization of methacrylic anhydride (MeAn), exhibiting either cyclopolymerization where two vinyl groups are consumed sequentially in the reaction, or a more conventional reaction where only one vinyl group reacts initially, and a second vinyl group reacts later to form a crosslink. [Figure 2] This is a reaction scheme demonstrating the reaction of a methacrylic anhydride-based polymer with a nucleophile (RXH, where X can be, for example, O, N, or S), which can take the form of hydrolysis or functionalization. The reaction of the anhydride to crosslink the two chains results in loss of crosslinking. [Figure 3] This figure shows three methods for forming micron-range microgel particles with amphoteric polymer electrolyte properties. All three methods begin with precipitation polymerization of a temporary divinyl crosslinking agent (e.g., methacrylic anhydride, MeAn) and a permanent crosslinking agent (e.g., diethylene glycol dimethacrylate (DEGDMA)). [Figure 4] This figure shows two methods for forming nanoparticles for use as antigen carriers in vaccine applications. Both methods involve initial precipitation polymerization of a transient crosslinking agent (e.g., methacrylic anhydride) and a gradually eroding divinyl crosslinking agent (e.g., disulfide-crosslinked dimethacrylate) to ensure that the particles are eventually removed by renal clearance. [Figure 5] This figure shows the formation and functionalization of a reactive particle platform composed of temporary and permanent crosslinking agents for generating microgels. [Figure 6-1] This figure shows optical microscope images of MeAN / DEGDMA(90:10) particles prepared in 60:40 MEK / heptane. Figure 6A - Post-formation in DMF (anhydrous remains unchanged); [Figure 6-2] Figure 6B - Hydrolyzed particles (anhydrous cleavage) in phosphate-buffered saline (PBS) (pH 2); and [Figure 6-3] Figure 6C - Hydrolyzed particles in PBS (pH 7.4). Size bar: 20 μm. [Figure 7]This figure shows bright-field optical microscope images of MeAN-only (MED-55 / 0 / 0) microspheres formed in 55 / 45 MEK / heptane in the absence of a permanent crosslinking agent. The particles were suspended in MEK and imaged. [Figure 8] Figures 8A-F show bright-field optical microscope images of MED-55 / 5 / 5 particles prepared using monomer concentrations of 2-7% in acetonitrile (8A: 2%, 8B: 3%, 8C: 4%, 8D: 5%, 8E: 6%, 8F: 7%). 100x oil immersion. Particles were dispersed in ACN and imaged. Size bar: 10 μm. [Figure 9] This graph shows the diameter as a function of the weight percentage of AIBN (initiator), clarifying the effect of the initiator content on the size of MED-62 / 0 / 10 particles produced by photopolymerization. [Figure 10] This graph shows the diameter as a function of MEK (vol%) in the solvent, revealing the effect of various MEK / heptane ratios on the size of MED-X / 0 / 10 particles produced by photopolymerization. The particles are formed with >62% MEK, but gradually become smaller, and their sizes (data points marked with "?") could not be accurately determined by optical microscopy. [Figure 11] This figure shows bright-field optical microscope images of anionic MED-55 microgels prepared with different ratios of EGDMA / DEGDMA permanent crosslinking agent (total 10 mol%). Top row - MED-55 / 10 / 0; Middle row - MED-55 / 5 / 5; Bottom row - MED-55 / 0 / 10. Scale bar = 5 μm; Image cropped for magnification. [Figure 12] This graph shows the swelling ratio as a function of pH for MED-55 / 10 / 0 (▲) and MED-55 / 5 / 5 (■), clarifying the effect of the crosslinking agent composition on the swelling of anionic microgels as a function of pH. The swelling ratio is normalized to the particle volume at pH 2.4 using (Dx / D2.4)³ [where Dx is the particle diameter at a given pH and D2.4 is the diameter at pH 2.4]. [Figure 13-1]Figure 13A: This figure shows an example of microgel particles formed by precipitating copolymerization of methacrylic anhydride (90 mol%) and DEGDMA (10 mol%) in a 60:40 MEK:heptane mixture with a total monomer content of 5 wt%, followed by functionalization with N,N-dimethylethylenediamine to produce amphoteric polymer electrolyte hydrogel particles. The particles are suspended in HEPES-buffered saline (pH 7.6). [Figure 13-2] Figure 13B: This graph shows the distribution of particle sizes expressed in units of μm² as particle area. Most particles have an area of ​​4 to 6 μm², corresponding to a particle diameter of 2.25 to 2.75 μm. [Figure 14] Figure 14A: Bright-field microscope image of MED-55 / 5 / 5 particles in DMF before hydrolysis. Figure 14B: Microscopic image of densely packed multilayer DMAPA and TAMRA-functionalized MED-55 / 5 / 5 particles in water. Figure 14C: Confocal fluorescence microscope image of DMAPA and TAMRA-functionalized MED-55 / 5 / 5 particles in water. Size bar: 15 μm. [Figure 15] This bar graph shows the zeta potential of MED-55 / 10 / 0 microspheres measured in PBS (pH 7.4) after hydrolysis and functionalization with DMAPA. [Figure 16] This figure shows the 1H-NMR (600 MHz) spectrum of (propane-2,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(2-methylacrylate) (KTMA) in CDCl3. [Figure 17] This figure shows an optical microscope image of MKT-55 / 15 particles prepared with 85:15 MeAn / KTMA in 55:45 MEK / heptane. Size bar: 5 μm. [Figure 18] This figure shows bright-field optical microscope images of MKT-55 / 15 anionic microgels at pH 5 (top row), pH 7 (middle row), and pH 10 (bottom row) over time. Scale bar = 15 μm. [Figure 19]This graph shows the cell viability immediately after thawing and the percentage of recovered 3T3 cells after a 24-hour freeze / thaw cycle in the presence of MED-55 / 2 / 8 10wt% (■), MED-55 / 2 / 8 5wt% (◆), a negative control (●), and DMSO 10v / v% (▲). [Figure 20] This graph shows the number of 3T3 cells per day after thawing for cells frozen with DMEM containing amphoteric polyelectrolyte microgel or DMSO, and for negative controls of cells frozen in DMEM without supplemental cryoprotection. MED-55 / 2 / 8 10wt% (■), MED-55 / 2 / 8 5wt% (◆), negative control (●), and DMSO 10v / v% (▲). [Figure 21] This figure shows a bright-field microscope image of 3T3 cells after freezing them with a cryoprotective amphoteric polymer electrolyte microgel and then thawing them. [Figure 22] This figure shows bright-field and fluorescence images of pDMAEA-grafted particles after exposure to fluorescein-labeled ovalbumin (OVA-FITC) in PBS (pH 7.4). Figure 22A: Bright-field image of pDMAEA-grafted particles in PBS (pH 7.33) with OVA-FITC, and Figure 22B: Fluorescence image of pDMAEA-grafted particles in PBS (pH 7.40) with OVA-FITC. [Figure 23] This figure shows confocal fluorescence images of fluorescently stained 3T3 cells co-cultured with TAMRA-labeled MED-55 / 5 / 5 microgel. Figure 23A - DMAPA amphoteric polyelectrolyte, Figure 23B - RGD anionic, Figure 23C - anionic. [Figure 24] This figure shows a confocal image of NIH 3T3 cells stained with calcein-AM mixed with TAMRA-labeled amphoteric polyelectrolyte MED-55 / 15 / 0 microgel. [Figure 25]The figures show confocal images of NIH 3T3 cells stained with calcein-AM and ethidium-homodimer LIVE / DEAD staining, after co-encapsulating PLL / PM50 calcium alginate capsules with MED-55 / 10 / 0 amphoteric polyelectrolyte microgel at a cell concentration of 2.0 × 10⁶ cells / mL and 0.5 wt / v% microgel (Figure 25A: scale bar 100 μm, and Figure 25B: scale bar 15 μm). [Modes for carrying out the invention]

[0077] The present invention includes several embodiments.

[0078] In one embodiment, a novel composition of monomers and crosslinkers for precipitation polymerization is provided, designed to enable the formation of a new type of polymer microgel particles in high yield through a scalable process, and to combine properties that have not been previously attainable. These properties include, but are not limited to, that the polymer microparticles are lightly crosslinked, swellable, narrowly dispersed or monodispersible, stabilizer-free, reactive, and optionally degradable. Such particles can serve as a platform for highly defined hydrogel particles for use in various areas of biomedicine.

[0079] In other embodiments, novel applications for nano and microgel particles are provided, including cryoprotection particles, granular ECM components, and charge-shift vaccine platforms.

[0080] In some embodiments, microgel particles are formed by a novel precipitation polymerization method taught herein.

[0081] As used herein, the term "microgel" refers to a lightly crosslinked polymer system in the form of microparticles that are swollen by a solvent. As used herein, the term "hydrogel" refers to a lightly crosslinked polymer system that is swollen in water.

[0082] As used herein, the term “microparticles” is generally used to refer to particles with a size of 1 to 1000 μm, but unless otherwise indicated in the context, it may also include submicron particles (i.e., nanoparticles within this size range) with a size of 0.1 to 1 μm. In preferred embodiments, the microparticles have a particle diameter of 0.1 to 50 μm, more preferably 0.3 to 30 μm, or even more preferably 0.5 to 20 μm.

[0083] As used herein in relation to polymer matrices, the term "covalently crosslinked" refers to the formation of covalent bonds between polymer chains that hold together the polymer matrix (microparticles in this study). Polymers cannot readily dissolve into individual polymer chains when covalent crosslinks are present. In the case of initially formed reactive particles, covalent crosslinks can be provided by either temporary or permanent crosslinking agents. After the temporary crosslinks are cleaved by hydrolysis or functionalization, the overall network structure of the hydrogel particles is maintained by covalent crosslinks provided by permanent crosslinking agents.

[0084] In some variations, the permanent crosslinker is a gradually degrading crosslinker that can be degraded under physiological conditions over a timeframe of approximately 2 hours to 2 weeks, preferably 8 hours to 48 hours. Such gradually degrading crosslinkers can be based on bisacrylate or bismethacrylate crosslinkers containing disulfide bonds that can be cleaved over time under physiological conditions by a reductive process involving a reaction with physiological glutathione, which is chemically orthogonal to the processes used to chemically modify or hydrolyze anhydrous-based transient crosslinkers, such as after administration to tissue during vaccination. Other variations are described below and include other bisacrylate or bismethacrylate crosslinkers containing other cleavable linkers between two acrylate or methacrylate units. The purpose of using such gradually degrading crosslinkers is that they allow for particle clearance after use.

[0085] As used herein, the term “amphoteric polymeric electrolyte” refers to a zwitterionic polymer comprising positively charged and negatively charged monomer units, where positive and negative charges appear in various monomer units. The amphoteric polymeric electrolyte discussed herein is a copolymer comprising anionic and cationic comonomers (and optionally neutral and hydrophobic comonomers) grafted via residual or introduced vinyl groups bonded to microgel particles. In one embodiment, the amphoteric polymeric electrolyte comprises cationic units that are primary amines and anionic units that are carboxylic acids. Generally, the amphoteric polymeric electrolyte may contain about 10–90 mol% positively charged monomers and 90–10 mol% negatively charged monomers, preferably about 30–70 mol% positively charged monomers and about 70–30 mol% negatively charged monomers.

[0086] The final particles may contain at least 10%, preferably 50-400%, of amphoteric polyelectrolytes by dry weight.

[0087] In other embodiments, the cationic group may be a monomer containing a secondary, tertiary, or quaternary ammonium group, or a monomer containing a guanidium group, or a monomer containing a sulfonium group, or a monomer containing a conjugated diazole group, such as those found in imidazoles and similar cyclic and linear groups known to those skilled in the art.

[0088] In other embodiments, the anionic group may consist of monomers containing a carboxylic acid group, such as acrylic acid or methacrylic acid, or precursors of such monomers, such as t-butyl acrylate or t-butyl methacrylate.

[0089] In further embodiments, the hydrolyzed microgel acts as the polyanionic component, and the grafted polymer or copolymer acts as the cationic component. In such embodiments, the cationic component may be a homopolymer containing a cationic monomer incorporating primary, secondary, tertiary, or quaternary cationic monomers based on acrylate, methacrylate, acrylamide, or methacrylamide polymerizable units. In related embodiments, the cationic component may be a copolymer containing one or more of the above cationic monomers together with a neutral or anionic comonomer. In such copolymers, the cationic monomer(s)

[0090] In further embodiments, the amphoteric polymer electrolyte microgels are formed by precipitation copolymerization of a temporary crosslinking agent, a cationic monomer, and a permanent crosslinking agent, or by precipitation copolymerization of a temporary crosslinking agent, a cationic monomer, and a gradually degradable disulfide-containing crosslinking agent. In these embodiments, the amounts of the temporary crosslinking agent and the cationic monomer are selected so that the final anionic / cationic ratio can be controlled from 10 / 90 anionic / cationic to 50 / 50 anionic / cationic. Suitable cationic monomers for this embodiment include tertiary amines such as 2-(dimethylamino)ethyl methacrylate and N-(3-(dimethylamino)propyl) methacrylamide.

[0091] In yet another embodiment, the cationic polymer or copolymer is not grafted through using residual or newly introduced vinyl groups, but rather introduced by electrostatic complex formation between the anionic hydrolyzed microgel particles and the soluble cationic polymer or copolymer. As is known to those skilled in the art, polyanionic polymers have a strong affinity for binding polycationic polymers to form polyelectrolyte complexes. This complex formation between the cationic or net cationic polymer and the anionic polymer network is driven by the associated release of small counterions from both charged polymers involved. Furthermore, it is known in the art that the resulting polyelectrolyte complex can have physical properties ranging from a solid precipitate of insoluble PEC to a liquid complex phase called a complex coacervate, depending on the net strength of the electrostatic interaction between the two charged polymers. As is further known in the art, polyanionic hydrogel particles have the ability to bind not only polycations consisting solely of cationic monomers (cationic homopolymers), but also cationic copolymers, which include mixtures of cationic monomers with hydrophilic, neutral, or even anionic comonomers. These copolymers are non-stoichiometric amphoteric electrolytes, defined as copolymers having an excess of cationic monomers compared to anionic monomers to enhance the absorption of amphoteric polymer electrolytes into anionic microgels. Such non-stoichiometric amphoteric electrolytes can contain 30-99% cationic monomers, preferably 50-80% cationic comonomers, most preferably 60-70% cationic comonomers. In addition to cationic and anionic monomers, they can also contain neutral and even hydrophobic comonomers. Cationic monomer (3-aminopropyl methacrylamide, APM) and anionic monomer methacrylic acid 6 Alternatively, a neutral hydrophilic comonomer N-(2-hydroxypropyl)methacrylamide (HPM) 7 This copolymer can be absorbed into calcium alginate hydrogel beads.

[0092] In some embodiments, the term “temporary crosslinker” as used herein refers to a crosslinker used to produce polymer particles that have not yet been functionalized. The temporary crosslinker is used to graft one or more functional groups, such as amines, carboxyls, or thiols, depending on the desired application of the polymer. The temporary crosslinker may have anhydride groups and may be a crosslinker of formula (I) described below. In one embodiment, the temporary crosslinker is completely cleaved. However, even when the temporary crosslinker is completely cleaved, the integrity of the polymer in the solvent can be maintained for the sake of a permanent crosslinker.

[0093] As defined herein, the term “permanent crosslinker” refers to a crosslinker that survives, largely unimpaired, the conditions used to hydrolyze or functionalize a temporary crosslinker, which is part of the same polymer microparticles. For example, the conditions may be those used to hydrolyze or functionalize anhydride groups. It may be one that can be cleaved under various conditions, or simply more slowly, as described below. In one embodiment, the permanent crosslinker has at least two vinyl groups. An example is a combination of methacrylic anhydride, which is unstable to hydrolysis, and mono, di, and higher ethylene glycol dimethacrylates, which are stable to hydrolysis. In some embodiments, the permanent crosslinker is a degradable or biodegradable crosslinker. Degradable or biodegradable crosslinkers do not react during the hydrolysis or functionalization reaction of the temporary crosslinker but degrade in vivo under physiological conditions. Such degradable crosslinkers include those containing ketals or disulfides that survive the hydrolytic cleavage of the temporary crosslinker but degrade under physiological conditions over several hours to several weeks.

[0094] As used herein, the term “functionalization” refers to a reaction in which a functional group is generated from a reactive group. The functional group may be a peptide group (e.g., RGD) or other molecule (e.g., fluorophores, polymers, etc.). Functionalization involves reactions with nucleophiles such as water (hydrolysis), amines, alcohols, and thiols to cleave the anhydride group of a temporary crosslinking agent. As an example, hydrophobic, hydrophilic, or bioactive groups having thiols can be used. Furthermore, the reaction may also involve a difunctional species such as a diamine. For example, a difunctional species is defined as a diamine in which both amines are primary or secondary amines, e.g., 1,2-ethylenediamine or 1,3-propylenediamine, and when involved in a functionalization reaction, if both ends of the diamine react with anhydride groups, the anhydride crosslink may be broken down and replaced with a diamide crosslink, and a new crosslink may be formed. Similarly, functionalization may require diamines, triamines, or higher amines, where only one amine group is a primary or secondary amine and the other amine groups are tertiary or quaternary amine (ammonium) groups. Examples include N,N-dimethylaminopropylamine, as well as similar diamines and higher amines known to those skilled in the art. A particular aspect of this functionalization with higher amines is the ability to introduce a cationic group in excess of anionic groups into the hydrogel particles.

[0095] As defined herein, the term “biocompatible” refers to a compound or microparticle that is compatible with long-term in vitro or in vivo contact with cells and / or specific biological tissues. Biocompatible compounds or microparticles do not induce significant negative effects on cell viability, cellular function, and / or tissue function, thereby biocompatibility is typically designated in that they are compatible with a specific tissue or cellular environment.

[0096] In one embodiment, the present invention provides swellable, stabilizer-free, reactive, narrow-size dispersible nano and microparticles in high yield that can be modified to serve as useful activators for various biomedical applications.

[0097] This comprises highly swellable microgel particles functionalized with amine and carboxylic acid units in a ratio of 80:20 to 20:80, preferably 70:30 to 30:70.

[0098] These microgel particles can also be functionalized by graft-through or graft-from using a mixture of anionic and cationic comonomers to again achieve an anionic-to-cation charge ratio of about 80:20 to 20:80, preferably 70:30 to 30:70.

[0099] Examples of neutral hydrophilic monomers for such functionalization include 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, acrylamide, methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-isopropylacrylamide, (and other acrylamide / methacrylamides), PEG methacrylate, N-vinylpyrrolidone, and similar monomers known to those skilled in the art.

[0100] Examples of neutral hydrophobic monomers include alkyl(C1-C12) methacrylates and acrylates, alkyl(C4-C12) methacrylamides and acrylamides, styrene, 4-methylstyrene, and other substituted styrenes.

[0101] Examples of anionic monomers include acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-acrylamido-2-methylpropanesulfonic acid (or sodium salt), vinyl sulfonic acid, styrene sulfonic acid (or sodium salt), vinyl-functional phosphoric acid, and phosphonic acid, including, but not limited to, vinylphosphonic acid and 2-(methacryloyloxy)ethyl phosphoric acid.

[0102] Examples of cationic monomers include N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminoethyl acrylate, 3-(N,N-dimethylamino)propyl methacrylamide, 3-aminopropyl methacrylamide, 2-(methacryloyloxyethyl)trimethylammonium chloride, 3-(methacrylamidopropyl)trimethylammonium chloride (all represented by the general structures shown below), and vinylpyridine.

[0103] [ka]

[0104] Examples of zwitterionic monomers include 2-methacryloyloxyethyl phosphorylcholine, N-(2-methacryloyloxy)ethyl-N,N-dimethylammoniopropanesulfonate, N-(3-methacryloylimino)propyl-N,N-dimethylammoniopropanesulfonate, 3-(2'-vinylpyridinio)propanesulfonate, and 3-[[2-(methacryloyloxy)ethyl]-dimethylammonio]propionate (CBMA).

[0105] In some embodiments, the polymer may be grafted with monomers after functionalization. The grafted polymer network may contain up to 50 mol%, preferably up to 20 mol%, of hydrophobic monomers, such as butyl acrylate. In some embodiments, the polymer network contains neutral hydrophilic comonomers designed to enhance desired properties. An example is the addition of monomers having carbohydrate groups to enhance cryoprotection properties. Examples of neutral hydrophilic monomers include 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, acrylamide, methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-isopropylacrylamide, (and other acrylamide / methacrylamides), and PEG methacrylate. Examples of neutral hydrophobic monomers include alkyl (C1-C12) methacrylates and acrylates, alkyl (C4-C12) methacrylamides and acrylamides, styrene, and 4-methylstyrene.

[0106] Prior art particle formation methods require the addition of stabilizers or surfactants (e.g., poly(vinyl alcohol), poly(vinylpyrrolidone), cellulose, sodium dodecyl sulfate, etc.) to the polymerization mixture to successfully form particles. Some stabilizers or surfactants added by known methods permanently bind to the particles or are very difficult to remove. In the context of stabilizers and surfactants, the terms “add” or “added” as used herein refer to the voluntary addition of stabilizers and / or surfactants to promote colloidal stability in the formation of microparticles. Microparticles precipitated according to the methods taught herein, without added stabilizers and / or surfactants, are described as “substantially free” of stabilizers or surfactants, but allow for the presence of minimal amounts of stabilizers (e.g., as impurities). In this precipitation polymerization method, no stabilizers or surfactants are added at any point in the process. Therefore, the particles do not contain added stabilizers and / or surfactants. In some embodiments, the surfactant and / or stabilizer is less than 0.1 wt% of the total monomer. Therefore, the surface of such precipitated polymer particles is defined solely by comonomers and crosslinking agents present at particle formation, which are any group of initiators used to initiate particle-forming polymerization of graft-through polymerization and any subsequent functionalization. In preferred embodiments, any stabilizer or surfactant is an impurity, i.e., added unintentionally. In one embodiment, the particles according to this disclosure contain less than 1% (unintentionally added) surfactant or stabilizer. Those skilled in the art will understand that 1 The composition of the particles can be determined according to methods known in the art, such as 1H nuclear magnetic resonance spectroscopy or other chemical techniques such as solidification.

[0107] The microparticles of the present invention are narrowly dispersed or monodisperse. In one embodiment, the microparticles have a size distribution with a coefficient of variation of less than about 0.3, less than about 0.2, or less than about 0.1.

[0108] Microparticles can be generated in a variety of different sizes while maintaining favorable narrow or monodispersion.

[0109] The average particle size can be varied while maintaining a narrow dispersion size distribution (coefficient of variation less than approximately 0.3) by adjusting various synthesis conditions, such as the properties of the solvent. The average diameter ranges from 0.2 to 20 μm, depending on the solvent composition. The solvent composition can be adjusted to obtain narrowly dispersed particles with diameters within this range. Within this entire range, particles with diameters in the 1 to 10 micrometer range are the easiest to achieve.

[0110] These microparticles can be characterized as swellable when their transient crosslinks are cleaved by hydrolysis or functionalization. In one embodiment, the microparticles have a wet-to-dry swelling ratio of about 3:1 to about 50:1. In some applications, including cryoprotection and cell chaperones, the wet-to-dry swelling ratio is preferably 5:1 to 30:1. In applications such as vaccine encapsulation, a wet-to-dry ratio of 3:1 to 10:1 is preferred. Thus, the swelling ability of the microparticles can be adjusted for the desired specific application. Particle swelling contributes to the colloidal stability of the particles by limiting the phenomenon of particles sticking together.

[0111] For biomaterial applications, lightly crosslinked, swollen hydrogels containing 0.1 to 30 mol%, preferably 2 to 10 mol%, of a permanent crosslinking agent relative to the total monomer are often desirable to better mimic tissue properties.

[0112] These microparticles can be characterized as flexible and mildly crosslinked. In one embodiment, the polymer of the microparticles contains 0.1 to 20 mol%, preferably 1 to 10 mol%, of a permanent crosslinking agent relative to the total monomers of the polymer. Functionally, "flexibility" can be defined as being similar to that of biological tissue in terms of deformability. Quantitatively, "flexibility" can be defined as having the same deformability as cells and tissues, which is about 100 Pascals (Pa) to about 100 kPa, about 5 to about 50 kPa, or about 1 to about 10 kPa.

[0113] The particles of this disclosure generally have a spherical shape with a smooth or rough surface. In one embodiment, the shape is spherical or irregular sphere. An irregular sphere can be defined as having small ridges on its surface, thereby making the surface rough. While we do not wish to adhere to any particular theory, the spherical or irregular sphere properties are explained in a manner in which particles are formed by precipitation polymerization, where the particles grow upon addition and thus proceed toward a spherical shape. This shape can be advantageous in that it promotes a densely packed arrangement, whether in other particles or cells.

[0114] Due to the properties detailed herein, the microparticles and the method of producing them can offer many advantages. The precipitation polymerization method described herein allows for control of the radial composition profile, including the composition profile and crosslinking density profile of microgel particles, enabling better management of microgel-cell interactions. A narrow size distribution can limit the deformation of mixed cells compared to irregular microparticles. While we do not wish to dwell on any particular theory, a narrow size distribution allows for the formation of densely packed arrangements of particles that give consistent void volumes between microgels. Irregular particles also have considerably smaller void volumes that can be densely packed together with varying void volumes, increasing the deformation of mixed cells. • Surfactants or stabilizers typically present on particles formed by other polymerization techniques can influence and potentially dominate cell-particle interactions. In contrast, the absence of any surfactants or stabilizers added to the microparticle surface, a discriminant feature of precipitation polymerization, means that cell-microparticle interactions are primarily driven by the chemical and biological groups present on the particle surface as a result of the selection of monomers, initiators, and functionalizing reagents during particle preparation. The precipitation polymerization method described herein enables high-throughput and high-yield production of microparticles. · These micro-particles can be efficiently post-modified to adjust their particle properties. Examples of functionalizing agents include molecules containing nucleophilic groups such as (primary or secondary) amines, hydroxyls, or thiols, such as those shown below.

[0115] [Chemical formula] [Here, A = H, alkyl (linear and branched, C1 - C 12 ), phenyl, benzyl, dialkylaminoalkyl - or trialkylammonioalkyl, alkoxyethyl, oligo(ethylene glycol)].

[0116] For the purpose of cell attachment, A can also be a cell-binding motif such as the RGD amino acid sequence, and a larger extracellular matrix component such as laminin. For the purposes of research and particle tracking, A can also be a fluorescent group such as fluorescein or rhodamine, or other groups known to those skilled in the art. Finally, these microparticles can be designed to alter their charge equilibrium, for example, by precipitation polymerization, post-functionalization, graft-through, and hydrolytic charge shift of cationic components introduced during polycation absorption. Examples include embodiments in which the cationic component includes charge-shift cationic monomers such as dimethylaminoethyl acrylate (DMAEA) or other monomers, as well as functional groups known in the art to undergo spontaneous hydrolysis of ester bonds under physiological conditions with half-lives of approximately several hours and several days. Such groups can also be introduced, for example, during post-functionalization with lithium salts of N,N-dimethylaminoethanol, into particles swelling in tetrahydrofuran or 1,4-dioxane, or similar solvents or solvent mixtures known in the art to be aprotic polar solvents. Such groups can also be introduced by graft-through or graft-from, combining the particles with charge-shift monomers such as dimethylaminoethyl acrylate, or other cationic, neutral, anionic, or hydrophobic monomers designed to achieve the desired overall charge equilibrium in itself or in the final particle. Such groups can also be introduced by electrostatic absorption of polymers containing dimethylaminoethyl acrylate itself or in combination with other cationic, neutral, anionic, and / or hydrophobic monomers.

[0117] The final charge equilibrium of microgel particles produced by such functionalization, grafting, or absorption of charge-shifting groups may contain the majority of the cationic charge of microgels designed for use in antigen binding for vaccine development, or may have a near-stoichiometric ratio of cationic charge to anionic charge of microgel particles designed for cell cryoprotection.

[0118] Precipitation polymerization There is growing interest in the use of monodisperse, swellable, stabilizer-free hydrogel particles in biomedical applications, and therefore, a high-yield method for preparing such particles is needed. This specification provides a high-yield method for producing monodisperse, swellable, stabilizer-free hydrogel particles suitable for use in biomedical applications, and advantageously, a method that enables the formation of hydrogel particles with a variety of properties (e.g., size, stiffness, composition, e.g., present chemical / biological parts, radial distribution of properties such as crosslinking density and composition). In one embodiment, the yield is defined as the weight or molar ratio of the starting monomer and optionally the initiator to the monomers present in the formed polymer. In another embodiment, the yield is defined as the weight or molar ratio of the starting monomer and optionally the initiator to the monomers present in the particles. In various embodiments, the yield can be at least 30%, at least 40%, at least 50%, at least 60%, preferably at least 70%, or at least 80%.

[0119] Hydrogel particles having the above characteristics can be advantageously obtained by precipitation polymerization of one or more reactive monomers under particle-forming conditions, followed by hydrolysis and / or functionalization of the reactive monomers using a suitable modifier, and then swelling in water.

[0120] Precipitation polymerization is well-suited for producing particles containing reactive monomers (one or more), particularly water-sensitive particles, and for generating narrowly dispersed, micron-sized particles free of stabilizers or surfactants. However, the relatively high levels of crosslinking agents required for efficient particle formation in precipitation polymerization tend to result in particles that are far stiffer than those suitable for most biomaterial applications.

[0121] Precipitation polymerization begins with a homogeneous solution of monomers, of which at least one is a crosslinking agent and initiator. As the polymer forms, it precipitates from the solution. As clearly shown in the examples, the particles of this disclosure are formed under the conditions described herein. These conditions typically include a total monomer content of about 1 to about 20 wt%, or about 2 to about 10 wt%, a crosslinking agent fraction (see total monomers) of about 10 to about 100 mol%, or about 20 to about 80 mol%, and most importantly, a solvent having appropriate dissolving properties for the formed polymer. Particle formation becomes inefficient and limited at a total monomer content of less than 1 wt%. In one embodiment, only the crosslinking agent is used in the proportion. In one embodiment, one or more additional monomers (not temporary or permanent crosslinking agents) can be added to the monomer proportion to produce polymers adapted for specific applications. In one embodiment, the ratio of temporary to permanent crosslinking agent is about 90:10 to about 80:20. The solvent should be poor enough to allow the polymer to aggregate and form particles, but good enough to allow the polymer chains on the particle surface to swell and prevent particle-to-particle aggregation during polymerization. In one embodiment, the solvent used is about 4 to about 5 MPa, which is higher or lower than the value of the polymer being formed. 1 / 2 It has a Hildebrand solubility parameter of (i.e., almost polar). For example, poly(divinylbenzene) by precipitation polymerization (19.3 MPa) 1 / 2 The formation of ) is carried out by the solvent acetonitrile (24.3 MPa 1 / 2 ) and 20:80 MEK / heptane (15.9MPa) 1 / 2) can be carried out in a manner to obtain monodisperse microparticles according to the present disclosure. Precipitation polymerization can be used to form particles from reactive monomers such as methacrylic anhydride (i.e., those that allow for subsequent functionalization of the particles). Furthermore, in some embodiments, the viscosity of the solvent is another factor to be considered in the selection of the 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 higher than the polymerization temperature (typically 60-70°C for thermally initiated polymerization) and should not substantially react with the monomer or initiator. In the case of reactive monomers such as MeAn, nucleophilic solvents such as water, alcohol or amine should be avoided. Particles can also be obtained from photoinitiated precipitation polymerization, which allows for the use of solvents with lower boiling points. Examples of solvents suitable for 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. For photoinitiated polymerization, solvents such as acetone, diethyl ether, dichloromethane, and pentane may be used.

[0122] Hydrogels required for cellular applications are typically highly hydrated, flexible, and correlate with a low degree of crosslinking within the gel. However, low levels of crosslinking agent during precipitation polymerization are associated with low particle yield.

[0123] The present invention describes the use of a reactive transient crosslinker during precipitation polymerization to increase particle yield to at least 30%, preferably at least 50%, and most preferably at least 70%, compared to the typical yield of less than 20% for prior precipitation polymerization methods using low amounts of crosslinker. Equally important, after polymerization is complete, the reactive crosslinks are cleaved, allowing for particle swelling. Simultaneously, this transformation allows for the introduction of hydrophilic ionic groups and additional desired functional groups through careful selection of the cleaving reagent. A moderate amount of permanent (which may be more gradually eroded) crosslinker (5-20 mol% relative to the transient crosslinker) is included to prevent complete dissolution of particles during post-polymerization modification. In short, the inventors have found, surprisingly, that by using precipitation polymerization, they can obtain in high yield particles with cleavable crosslinks that can be readily functionalized and easily reach highly hydrated, flexible, narrowly dispersed microgels.

[0124] It has been found that monomers such as methacrylic anhydride (MeAn) or its acrylic acid analog, acrylic anhydride, are particularly suitable for this method because they can produce polymers having readily functionalized anhydride groups, and the anhydride crosslinks can be easily cleaved, allowing the as-formed highly crosslinked particles to swell into microgel particles.

[0125] MeAn is a divinyl monomer that undergoes two types of polymerization, crosslinking and cyclopolymerization (non-crosslinking polymerization), consuming both vinyl groups (Scheme 1). While not wishing to dwell on specific theories, cyclopolymerization is a "linear" polymerization in that it does not result in branching or crosslinking. In cyclopolymerization, the growing polymer chain adds two vinyl groups, alternately forming rings (5-membered or 6-membered rings in the case of MeAn) along the single polymer chain. Even though both vinyl groups are consumed, it is not crosslinking. Some divinyl monomers, such as diallyldimethylammonium chloride, experience only cyclopolymerization, while MeAn exhibits both types of reactions in varying ratios depending on experimental conditions (temperature, solvent, monomer concentration). 8

[0126] Therefore, equations (I), (IIa)~(IIf)

[0127] [ka] The present invention provides a temporary crosslinking monomer of the formula [wherein R1 and R2 are independently selected from H, a C1-C4 linear or branched carbon chain, benzyl, phenyl, or OJ, and J is defined as a C1-C4 linear or branched carbon chain].

[0128] [ka]

[0129] The symmetrical and mixed anhydrides (shown in formulas IIa to IIf) are suitable temporary crosslinking agents. Compound IIa is 4-vinylbenzoic anhydride, compound IIb is 3,4-vinylbenzoic anhydride, and compound IIC is 3-vinylbenzoic anhydride. All three formulas are suitable temporary crosslinking agents, as are mixtures of different symmetrical or mixed anhydrides.

[0130] [ka] In the formula, n is an integer between 1 and 3.

[0131] [ka] In the formula, R3 is independently either H or methyl.

[0132] Cyclic anhydrides such as maleic anhydride, citraconic anhydride, or itaconic anhydride have only one vinyl group and cannot be considered as crosslinking agents; therefore, they are not suitable as temporary crosslinking agents for this method. Furthermore, these compounds exhibit insufficient polymerization efficiency under certain conditions, for example, when present as monomer mixtures at concentrations higher than 50 mol%, which limits their usefulness.

[0133] Figure 1 illustrates the polymerization of methacrylic anhydride, showing cyclopolymerization in which two vinyl groups are consumed sequentially, or a more conventional reaction in which only one vinyl group reacts. When the second vinyl group reacts later, a temporary crosslink is formed.

[0134] The formed precipitated microparticles are of formulas (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), (IIIn), and / or (IIIo).

[0135] [ka] [In the formula, R1 and R2 are independently selected from H, a C1-C4 linear or branched carbon chain, benzyl, phenyl, or OJ, and J is defined as a C1-C4 linear or branched carbon chain. The dashed line represents an extended polymer backbone.]

[0136] [ka]

[0137] [ka] [In the formula, n is an integer between 1 and 3].

[0138] [ka]

[0139] [ka] The formula has a temporary crosslinking agent monomer [wherein R3 is independently H or methyl].

[0140] These monomers can be functionalized with, for example, a number of monomers of formula (IV) described below.

[0141] As illustrated in Figure 2, the polymer has MeAn groups in which only one vinyl bond has reacted, and in which both are consumed by cyclopolymerization or crosslinking. When the polymer is exposed to a nucleophile such as water, alcohol, thiol, or amine, the anhydride groups are consumed to form esters or amides, including carboxylic acids and thioesters. In the course of this reaction, the polymer is functionalized and the anhydride crosslinks are cleaved. If these are the only crosslinks, the particles dissolve. For these reasons, common crosslinking agents, such as ethylene glycol diacrylate, ethylene glycol dimethacrylate (EGDMA), diethylene glycol diacrylate, diethylene glycol dimethacrylate (DEGDMA), oligo(ethylene glycol) diacrylate, oligo(ethylene glycol) dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, N,N'-methylenebisacrylamide (MBA), N,N'-methylenedimethacrylamide, glycol, glycerol, pentaerythritol, polyvinyl or polyallyl ethers of carbohydrates; divinylbenzene (DVB), trivinylbenzene, divinylpyridine, or similar are added as permanent crosslinking agents in amounts of 1-30%, preferably 5-20%, of the total monomer weight to ensure that the particles survive functionalization and hydrolysis. The following scheme shows additional amine-functionalized permanent crosslinking agents, where R and R1 = H or methyl, R' = alkyl or aryl, and x = 1 or 2.

[0142] [ka]

[0143] If the microgel particles are required to undergo final spontaneous hydrolysis or enzymatic degradation, the permanent crosslinking agent can be selected from a known group of degradable crosslinking agents containing groups that can be cleaved over a suitable timeframe under physiological conditions, including, but not limited to, disulfide groups, unstable esters, unstable acetals and ketals, and hindered anhydride groups. They all take the form of degradable spacers between two monomer units. Degradation may occur in several ways, including hydrolysis, enzymatic, redox, or photochemical methods.

[0144] Examples of such gradually degradable crosslinking agents include divinyl or higher vinyl crosslinking agents containing disulfides, such as bis(2-methacryloyl)oxyethyl disulfide or bis(2-acryloyl)oxyethyl disulfide, or the corresponding methacrylamide or acrylamide (R1=Me, H; x=1~3).

[0145] [ka] Diacrylates or dimethacrylates containing unstable acetals, such as bis[(2-methacryloyloxy)ethoxymethyl]ether

[0146] [ka] Alternatively, a corresponding crosslinking agent containing a single acetal unit [wherein R1 = H, Me, and R2 = H, alkyl (C1-C8, linear or branched), or aryl].

[0147] [ka] These are some examples.

[0148] Other degradable crosslinking agents include bis-methacrylate-terminated polylactic acid / glycolic acid oligomers and their multi-arm analogs, as well as acrylate and acrylamide analogs [wherein R1=H, Me; R2=H, Me; R3=H, Me].

[0149] [ka] These are some examples.

[0150] Other degradable crosslinking agents, similar to crosslinking agents that hold acrylic acid esters based on a 2-aminoethanol motif, can be based on ester bonds that become degradable by proximity to amine groups, such as the two bisacryloylaminoesters shown below.

[0151] [ka]

[0152] Furthermore, the degradable crosslinking agents, as with similar bisacrylate, bisacrylamide, and bismethacrylamide crosslinking agents, and similar crosslinking agents containing longer spacers than ethyl, multiple dialkylsiloxane unstable units, and the multi-arm analogs described above, can incorporate hydrolysis-unstable siloxane bonds such as dimethyldi(methacryloyloxy-1-ethoxy)silane, as shown below.

[0153] [ka]

[0154] Furthermore, the degradable crosslinking agent may contain unstable ON bonds such as N,O-dimethacryloylhydroxylamine, as shown below.

[0155] [ka]

[0156] Finally, degradable crosslinking agents may contain matrix metalloproteinase (MMP) cleavable groups, such as (Pro-Leu-Gly-Leu-Trp-Ala), allowing matrix metalloproteinases (MMP1, MMP3, MMP7, and MMP9) to degrade the polymer network.

[0157] Figure 2 illustrates a reaction scheme showing the reaction of a methacrylic anhydride-based polymer with a nucleophile (RXH), which can take the form of hydrolysis or functionalization. The reaction of the anhydride to crosslink the two chains results in loss of crosslinking. X is selected from NH, NR, O, and S, and R is a suitable biocompatible compound or molecule. For example, R is linear or branched C1-C 18 These can be defined as aryls, heteroaromatics, sugars, fluorophores, amino acids, peptides such as RGD, polymerization initiators, polyethylene glycol (PEG), betaine, proteins, ethylenediamines, or other biomolecules (such as nucleotides, DNA, RNA, therapeutic molecules, or activators). Microparticles containing anhydrous monomers of formulas (IIIa) to (IIIc) are functionalized to become monomers of formulas (IVa) to (IVc). Similar monomers are derived from formulas (IIId) and (IIIe). The exemplary monomers shown below are derived from the hydrolysis and / or functionalization of transient crosslinking monomers of formulas (IIIa) to (IIIc).

[0158] [ka] [In the formula, R1 and R2 are H, alkyl (C1-C4), phenyl, or benzyl, X is O, NH, NR3, or S, and R3 is H, alkyl (C1-C4) 12 ), aryl, heteroaromatic, polyethylene glycol, sugar, fluorophore, amino acid, peptide, other biomolecules (such as DNA and RNA), or other monomers.

[0159] The microparticles described in this invention are formed by the precipitation polymerization of a specific mixture of comonomer and crosslinking agent under solvent conditions, and the resulting polymer takes the form of narrowly dispersed or monodisperse microspheres, with an average diameter ranging from about 0.3 to about 20 micrometers, depending on the properties and amounts of the solvent, monomer and crosslinking agent used.

[0160] Comonomers can be used together with crosslinking agents in the precipitation polymerization process. In one embodiment, the comonomer is alkyl (C1-C 12 ) Methacrylates and acrylates, alkyl (C4~C 12 ) These can be methacrylamide and acrylamide, styrene, 3- or 4-alkylstyrene [wherein alkyl can be linear or branched C1-C8], and styrene having alkyl ether or alkyl ester substituents at the 3rd and / or 4th positions.

[0161] Comonomers can also be acrylic and methacrylic monomers having ethylene glycol and methoxyethylene glycol side chains incorporating 1 to 4 units, and optionally mixed-length oligoethylene glycol side chains, as well as mixtures thereof.

[0162] [ka] [In the formula, R1 = H, alkyl (C1-C4), and R2 or R3 = H, alkyl (linear and branched, C1-C] 12 ), phenyl, benzyl, dialkylaminoethyl, dialkylaminopropyl, dialkylaminobutyl, alkoxyethyl, oligo(ethylene glycol), methoxyoligo(ethylene glycol).

[0163] The comonomer can also be vinyl ether, linear and cyclic N-vinylamide, or vinyl ester, for example, as shown below.

[0164] [ka] [In the formula, R1 = alkyl (linear or branched, C1-C8), X = O or NH, R2 = H, alkyl, or phenyl, and n = 1 or 2].

[0165] In one embodiment, the composition obtained on a dry weight basis contains microparticles ranging from 0.3 to 20 micrometers, with an average particle diameter (D) of less than 0.3, more preferably less than 0.2, or most preferably less than 0.1, in terms of the coefficient of variation. In one embodiment, the composition obtained on a dry weight basis contains microparticles ranging from 0.3 to 20 micrometers, with an average particle diameter of less than about 0.3, less than about 0.2, or less than about 0.1, in terms of the coefficient of variation. In one embodiment, the expression "contains microparticles" in the context of a composition is defined as a composition containing at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, or at least about 90% by weight of microparticles. The particle size can be selected on a specific application basis, such as whether or not the particles are to be taken up by cells; for example, a size of 0.1 to 1 micrometer is preferred for vaccine delivery, and a size of 1 to 10 micrometers is preferred for cryopreservation or cell chaperones.

[0166] One aspect of the present invention is that these nano and microparticles are formed in the presence of a high amount of transient crosslinking agent, which helps to achieve a high particle yield, and that subsequent controlled decrosslinking and chemical modification by hydrolysis and introduction of desired functional groups can be useful for several applications described below. Functionalization makes the nano and microparticles biocompatible. When in contact with aqueous bodily fluids, anhydrous groups react, lowering the pH and therefore not particularly biocompatible.

[0167] Figure 3 shows three methods for forming micron-range microgel particles with amphoteric polyelectrolyte properties. All three methods begin with precipitation polymerization with a temporary divinyl crosslinker (e.g., methacrylic anhydride, MeAn) and a permanent crosslinker (e.g., diethylene glycol dimethacrylate (DEGDMA)). In the first method, the temporary crosslinker (temporary XL) and the permanent crosslinker (permanent XL) undergo precipitation polymerization 101 to obtain high-density reactive microspheres 102. The high-density reactive microspheres are then modified with di or triamines 103 to form an amphoteric polyelectrolyte gel. In the second method, the high-density reactive microspheres 102 are 104 is hydrolyzed or functionalized to a vinyl-functionalized microgel. 105 is then grafted with cationic and anionic monomers to form an amphoteric polymer electrolyte microgel. In a third method, temporary and permanent crosslinking agents are combined with additional monomers in precipitation polymerization 106 to obtain high-density reactive microspheres 107. The high-density reactive microspheres 107 containing the additional monomers are then modified with di or triamines 108, or hydrolyzed to form an amphoteric polymer electrolyte microgel.

[0168] In one embodiment, microparticles formed by precipitation polymerization consist of or are essentially composed of a temporary crosslinking agent and a permanent crosslinking agent according to the Disclosure. In another embodiment, microparticles consist of or are essentially composed of methacrylic anhydride and / or acrylic anhydride and a permanent crosslinking agent according to the Disclosure. In yet another embodiment, the microparticles consist of or are essentially composed of methacrylic anhydride and / or acrylic anhydride and a permanent crosslinking agent selected from the group consisting of ethylene glycol diacrylate, ethylene glycol dimethacrylate (EGDMA), diethylene glycol diacrylate, diethylene glycol dimethacrylate (DEGDMA), oligo(ethylene glycol) diacrylate, oligo(ethylene glycol) dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, N,N'-methylenebisacrylamide (MBA), N,N'-methylenedimethacrylamide, glycol, glycerol, pentaerythritol, polyvinyl or polyallyl ethers of carbohydrates; divinylbenzene (DVB), trivinylbenzene, divinylpyridine, and combinations thereof. In an additional embodiment, the microparticles consist of or are essentially composed of methacrylic anhydride and / or acrylic anhydride and DEGDMA.

[0169] While we do not wish to adhere to any particular theory, the formation of microparticles is such that the density is higher in the core than on the surface, and the stiffness of the microparticles is higher in the core than on the surface. The particles grow by the deposition of newly formed polymers consisting of lightly crosslinked or branched polymer chains. The divinyl temporary crosslinkers of this disclosure provide more double bonds available for crosslinking than monovinyl monomers, and as the particles grow, there are more double bonds available to sustain the growth. Materials that are captured early in particle growth and therefore closer to the particle core undergo further crosslinking reactions, resulting in higher density and stiffness. The radial gradient can be further enhanced by using crosslinkers that are preferentially incorporated. For example, when a small amount of electron-rich crosslinker, such as divinylbenzene or divinyl ether, is added to a MeAn / DEGDMA polymerization, the electron-rich crosslinker is preferentially incorporated into the core due to its higher reactivity compared to other monomers, both of which are electron-deficient. This results in a higher level of electron-rich permanent crosslinker in the particle core than on the surface. These properties are inherent to the precipitation polymerization method used herein, as the sequential radial growth of particles results in a surface composition at some point in time, reflecting the composition of the copolymer formed at that time.

[0170] As an example, a cleavable temporary crosslinking agent such as methacrylic anhydride (MeAn) is copolymerized with a permanent crosslinking agent such as diethylene glycol dimethacrylate (DEGDMA) at a total monomer content of 5% (weight / volume) in the presence of approximately 2 wt% AIBN (relative to the total monomer) which serves as a radical initiator, in a solvent mixture containing 60 vol% methyl ethyl ketone and 40 vol% n-heptane. Polymerization is carried out in a 20 mL screw-top glass vial and heated at a temperature of 55–80°C, preferably 65–75°C, for 4–24 hours, preferably 12–20 hours. As is known in the art, there are thermal initiators that can initiate polymerization at lower and higher temperatures. Similarly, redox process-based radical initiators that can initiate polymerization at temperatures such as those incorporated herein are known in the art.

[0171] The ratio of temporary crosslinking agent to permanent crosslinking agent may range from 50:50 to 99:1 mol%, preferably from 80:20 to 95:5. Examples of reactive temporary crosslinking agents (formulas (I) and (II)) include MeAn, acrylic anhydride, and 4-vinylbenzoic anhydride. Examples of permanent crosslinking agents include EGDMA, DEGDMA, methylenebisacrylamide, and divinylbenzene.

[0172] In this example, the resulting microspheres are formed in high yield (67 ± 10%) because all monomers present during precipitation polymerization are divinyl compounds, resulting in a higher degree of crosslinking and therefore more efficient particle formation. This characteristic significantly increases the isolation yield (40% to 80%) of these microspheres compared to the isolation yield of comparable particles formed by precipitation copolymerization using the same amount of permanent crosslinking agent, but in the absence of a temporary crosslinking agent. In specific examples, the yield obtained by precipitation polymerization using a permanent crosslinking agent to temporary crosslinking agent ratio of 10:90 according to this disclosure is higher than the yield obtained by precipitation polymerization using a permanent crosslinking agent to simple monovinyl compound (non-crosslinking agent) ratio of approximately 10:90.

[0173] In some embodiments, an initiator, preferably a photoinitiator, can be used to decouple the rate of initiation from the reaction temperature. Both the polymerization rate and the polymer radial distribution are influenced by the reaction temperature. By using a photoinitiator instead of a thermal initiator, a constant rate of initiation can be achieved over a range of reaction temperatures. The photoinitiator can be selected such that the wavelength of the photostimulus is not absorbed by the solvent or monomer. For example, 2,2'-azobis(2-methylpropionitrile) (AIBN) can be used to initiate precipitation polymerization after irradiation with light having a wavelength of 365 nm.

[0174] cryopreservation Swelled and suspended microgels having the properties described herein may be combined with mammalian cells and other cells during culture, preferably in a microgel-to-cell volume ratio of 10,000:1 to 1:1, more preferably 5,000:1 to 200:1. In some embodiments, the microgels form a densely packed gel after co-precipitation with cells. The densely packed gel is a densely packed array of soft particles that are solid-like or gel-like under low stress but can flow under higher stress. Cell capture within the densely packed gel can reduce freeze damage to cells by reducing pericellular ice crystal formation and partial dehydration of cells. In another embodiment, a solution or suspension of a highly swollen microgel forms a viscous solution that prevents cell sedimentation. In some embodiments, the microgels used for cryopreservation have an anionic:cationic ratio of 70:30 to 30:70. In some embodiments, the hydrogel microparticles have a concentration of 1 to 25 wt / v%. This value may depend on the microgel stiffness. A more rigid microgel that can co-precipitate with cells to form a densely packed gel can be effectively used in the range of 1-5 wt / v%, while a flexible microgel that can form a volume-filled viscous solution that prevents cell sedimentation in the concentration range of 5-25 wt / v% can be separated from cells by centrifugation.

[0175] During freezing and thawing, these microgels surround the cells, preventing ice crystals from penetrating the cell wall and causing cytotoxicity. Simultaneously, slow freezing of the continuous medium causes an increase in osmotic pressure in the microgel, resulting in partial dehydration of the cytosol. The resulting higher osmotic pressure (higher protein concentration) in the cytosol reduces ice crystal formation within the cells. Additionally, microgels can reduce ice crystal recrystallization under thawing conditions that would otherwise produce larger cytotoxic ice crystals. In another embodiment, the addition of microgels under rapid freezing conditions leads to permeation of the continuous medium, inhibiting ice crystal formation.

[0176] Due to their relatively large size (1-10 micrometers) and non-fouling properties, these microgels are unlikely to be taken up by mammalian cells through affinity-mediated processes or further phagocytic effects, and therefore are not suitable for use with cell cryoprotective agents such as dimethyl sulfoxide (DMSO) (including stem cells), as well as ethylene glycol / glycerin and other sugar-derived molecules. 9 Overcoming key concerns (with use in conjunction with cryopreserved blood), namely residual cytotoxicity and the effect of cryoprotective agents on the ability of cells to differentiate (into stem cell properties), as well as the time required to remove intracellular cryoprotective agents.

[0177] Monodisperse microgel particles allow for better control over the degree of deformation to the intercellular space volume between cells within the microgel. Furthermore, in some embodiments, the flexible, deformable amphoteric polyelectrolyte microgels of this disclosure can be used to replace conventional cryoprotective agents that are cell-permeable, such as DMSO. This is particularly advantageous for cells that are sensitive to cryoprotective cell-permeable agents (e.g., DMSO). The amphoteric polyelectrolyte microgels of this disclosure can prevent rapid cell sedimentation to ensure cell survival during the freeze-thaw process encountered during cryopreservation. Avoiding the formation of external ice crystals is important because these ice crystals can penetrate the cell membrane. Advantageously, external ice crystals can be minimized or avoided using these microgels. Furthermore, the microgels of this disclosure can also dehydrate the cytosol, thereby preventing cell damage due to intracellular ice crystal formation.

[0178] These cell-sized hydrogel particles are far less likely to be taken up by cells than linear polymers of similar composition, reducing concerns such as cytotoxicity or interference with cell differentiation. Furthermore, the microgels of this disclosure are formed from polymers that are sufficiently crosslinked to minimize or prevent cell uptake.

[0179] Method for preparing an impermeable cell cryoprotective agent The microspheres described herein can be converted into cryoprotected hydrogel microparticles by three methods.

[0180] Post-modification of the as-formed microspheres by treatment with a slightly excess of N,N-dimethylethylenediamine, 3-(dimethylamino)propylamine, N,N-dimethylaminoethanol, choline, amino acids, betaine, diamine, triamine, polyamine, or other similar compounds that convert the anhydride group into a combination of a free carboxylic acid and an amide or ester having an amine group. This modification essentially converts the transient anhydride crosslinks and cyclic anhydrides formed during precipitation polymerization into a mixture of anionic carboxylates and cationic groups, conferring amphoteric polyelectrolyte properties to the resulting microgels. As a result of the cleavage of the transient anhydride crosslinkers, and depending on the amount of permanent crosslinkers present, these microgels are then highly swellable and deformable, with a total modulus approaching that of mammalian cells and tissues. These particles have no or very minimal exogenous surface residues of stabilizers. These microgels have shown usefulness as non-cell-permeable cytoprotective agents.

[0181] An alternative method for forming monodisperse cryoprotective microgels involves grafting amphoteric polymer electrolytes onto hydrogel microparticles using suspended vinyl groups. These suspended vinyl groups can be residual vinyl groups from permanent crosslinking agents (e.g., DEGDMA) used in precipitation polymerization, or vinyl groups added by functionalization of reactive microparticles with, for example, 3-aminopropyl methacrylamide, 2-aminoethyl methacrylate, or 2-hydroxyethyl methacrylate. Hydrolysis of the as-form microspheres under weakly alkaline conditions, or hydrolysis following functionalization, converts the transient anhydride crosslinks and cyclic anhydride groups and suspended anhydrides to carboxylic acids, or in the case of functionalization, to amides or esters (functionalized carboxylic acids). Subsequently, the resulting highly swollen microgel can be modified into an amphoteric polymer electrolyte microgel by a process called graft-through. Hydrolyzable microgels are suspended in aqueous mixtures of anionic and cationic monomers, including but not limited to methacrylic acid (MAA) and N,N-dimethylaminoethyl methacrylate (DMAEMA), with a water-soluble radical initiator. The mixture is then heated or irradiated with light, resulting in copolymers of water-soluble monomers covalently bonded by grafting suspended vinyl groups. Examples of anionic, cationic, and zwitterionic monomers are listed below. Anionic acrylic acid, methacrylic acid, 2-carboxyethyl acrylate, 2-acrylamido-2-methylpropanesulfonic acid (or sodium salt), vinyl sulfonic acid, styrene sulfonic acid (or sodium salt), phosphonic acid. Cationic N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminoethyl acrylate, 3-(N,N-dimethylamino)propyl methacrylamide, 3-aminopropyl methacrylamide, 2-(methacryloyloxyethyl)trimethylammonium chloride, 3-(methacrylamidopropyl)trimethylammonium chloride (the preceding monomer is represented by the general structure shown below), and vinylpyridine.

[0182] [ka] Zwitterionic-2-methacryloyloxyethyl phosphorylcholine, N-(2-methacryloyloxy)ethyl-N,N-dimethylammoniopropanesulfonate, N-(3-methacryloylimino)propyl-N,N-dimethylammoniopropanesulfonate, 3-(2'-vinylpyridinio)propanesulfonate, 3-[[2-(methacryloyloxy)ethyl]-dimethylammonio]propionate, and other betaine monomers.

[0183] Finally, the as-formed microgels can be modified into impermeable cryoprotective microgels by hydrolysis followed by absorption of a net positively charged polycation or copolymer. Examples include homopolymers of permanent cationic monomers such as aminoethyl methacrylamide, dimethylaminoethyl methacrylate, and similar cationic monomers; homopolymers of dimethylaminoethyl acrylate and similar charge-shift monomers; and copolymers of such permanent or charge-shift monomers with other cationic, neutral, anionic, or hydrophobic monomers as described elsewhere herein. However, the resulting copolymers have a net cationic charge and contain 30-99 mol% of cationic monomers, preferably 50-80%, and most preferably 60-70%.

[0184] Provided that the above-described final microgels have an appropriate anionic-to-cationic group ratio, including ratios ranging from 80:20 to 20:80 anionic-to-cationic, preferably 70:30 to 40:60 anionic-to-cationic, the combination of these microgels in the form of a high-density suspension with (mammalian) cells has a swollen microgel-to-cell volume ratio in the range of 10,000:1 to 1:1, preferably 5,000:1 to 200:1, and has the ability to alter the freezing behavior of the water surrounding these cells, for example, preventing damage to these cells during long-term storage under cryogenic conditions. These microgels may optionally contain neutral hydrophilic and hydrophobic groups, which can be introduced during the original precipitation polymerization, post-functionalization at low molecular weight, post-grafting via polymerization, or, overwhelmingly, absorption after hydrolysis of cationic comonomers. Examples of neutral hydrophilic and hydrophobic monomers are described below. • Neutral hydrophilic functionalizing reagents - aminoethanol, PEG-amine, ethylene glycol, and glycine methyl ester (and other amino acids). Neutral hydrophilic monomers - 2-hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, acrylamide, methacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, N-isopropylacrylamide, (and other acrylamide / methacrylamides), and PEG methacrylate. Neutral hydrophobic functionalizing agents - alkyl (C4-C12) and arylamines, alcohols (C2-C12), and thiols (C2-C12). Neutral hydrophobic monomers - alkyl (C1-C12) methacrylates and acrylates, alkyl (C4-C12) methacrylamides and acrylamides, styrene, and 4-methylstyrene.

[0185] A key advantage of these microparticles and methods is that, due to their size and non-contaminating composition, the formed microgels are extremely unlikely to enter the cytosol of cryoprotected mammalian cells, thus eliminating a significant concern regarding the use of commonly used permeable cryoprotective agents, including ethylene glycol, propylene glycol, and dimethyl sulfoxide (DMSO), which are known sensitizers and cytotoxic molecules that have been shown to affect the ability of stem cells to differentiate.

[0186] Furthermore, these microparticles are expected to be less susceptible to cellular permeability than linear copolymers having similar compositions.

[0187] Another advantage is that when these microgels are formed by the precipitation polymerization method described herein, they are formed without the need for surfactants or steric stabilizers, and therefore the surface is free from or substantially free from these potentially interfering compounds.

[0188] Similarly, the continuous growth of particles through the process of precipitation copolymerization means that the outer shell is less cross-linked than the particle core and therefore more flexible.

[0189] Furthermore, these microgel particles can be separated from cells by simple centrifugation after thawing from cryopreservation, based on their different sedimentation rates relative to the cells. Alternatively, the microgels may be separated from the cells by filtration if the microgels are significantly smaller than the cells. An example would be a microgel with a diameter of 0.5–3 micrometers compared to a typical cell diameter of 10–15 micrometers.

[0190] Similarly, the cryopreservation and separation from cells after thawing of these microgels can be facilitated by incorporating magnetic nanoparticles into the microgel particles by trapping preformed magnetic nanoparticles during precipitation polymerization, or by forming magnetic nanoparticles within anionic hydrolyzed microgel particles prior to the uptake of cationic charges, using methods known in the art for forming such magnetic nanoparticles within anionic polymer gels. These methods involve absorption of a soluble iron salt followed by precipitation into insoluble magnetic iron oxide nanoparticles.

[0191] Finally, the described precipitation polymerization method, which includes high levels of transient crosslinking agents, yields flexible, well-hydrated, monodisperse or narrow-disperse microgels in high yield, enabling excellent packing around dispersed cells.

[0192] cytomimiles In this specification, we also provide similar nano and microparticles modified to serve as synthetic granular components of ECM for cells in different forms of cell cultures, but not limited to, for cell encapsulation for research or therapeutic purposes.

[0193] Such microgels can be used as cytomimiles in cell cultures of adherent cells, and can be used as granular components of synthetic complex organoids containing cells and microgels in a ratio of 1:100 to 1:1, preferably 1:20 to 1:3.

[0194] Such complexes can provide many of the cellular benefits offered by real organoids consisting of individual cells, including adhesion, without requiring additional nutrients and oxygen. In fact, the presence of these permeable microgels can increase the supply of nutrients and oxygen to cells located within the complex cluster, as the network of highly swollen, permeable microgels can act as an oxygen and nutrient diffusion pathway, and can also increase the efflux of low to medium molecular weight cell products, such as insulin.

[0195] In addition to helping maintain higher oxygen partial pressure and nutrient levels near therapeutic cells, such artificial diffusion pathways / networks in permeable microgels also have the potential to increase soluble signaling entering and leaving clusters, and therefore increase the kinetics of glucose-induced insulin responses, such as feedback regulatory systems.

[0196] In reaggregations of pancreatic islet cells or β-cells or other therapeutic cells having such microgel-based cytomimiles, these diffusion pathways can replace the extensive microvessels typically present in the islets of Langerhans.

[0197] These advantages apply particularly to functional therapeutic cells and organoids encapsulated in semipermeable hydrogel capsules for transplantation into humans as part of cell therapy for endocrine disorders such as diabetes, hemophilia, and lysosomal storage disorders.

[0198] Granular ECM components inside the capsule Microgels similar to those described above for use in cryopreservation are optionally further modified with cell adherent groups such as RGD (arginine-glycine-aspartic acid) and can be used as granular ECM in various types of cell culture applications. These include encapsulation with donor or stem cell-derived mammalian endocrine cells designed for cell therapy of endocrine disorders such as diabetes, Parkinson's disease, hemophilia, and lysosomal storage disorders.

[0199] These may also include cell cultures used to study cellular behavior in simulated tissues, including cancer cell migration through tissues during metastasis, immune cell migration as part of innate immune surveillance mechanisms within tissues, cross-migration of germ cells and maternal cells during placental formation in pregnancy, and, for example, the spread of bacterial infections within tissues.

[0200] Post-modification can be designed to introduce chemical properties that enable the use of microgel particles in several biomaterial applications.

[0201] Suitable microgels can be formed, for example, by precipitation polymerization of methacrylic anhydride and diethylene glycol dimethacrylate (DEGDMA) in a molar ratio of 90:10 (preferably 99:1 to 80:20, 95:5 to 85:15) in a methyl ethyl ketone / heptane mixture (60:40) in the presence of 2% by weight of AIBN, with a total monomer content of 5 wt% (preferably 1 to 20%, 2 to 10 wt%).

[0202] The resulting particles can be modified by hydrolysis or functionalization using a variety of modifiers, including amines, alcohols, and thiols having hydrophobic, hydrophilic, or bioactive groups. More specifically, these modifiers can be ammonia, or primary amines such as alkylamines [where alkyl can be methyl, ethyl, propyl, butyl, etc.], glucosamine, and ethanolamine.

[0203] The above microgels can be post-modified with cell adhesion molecules that include all protein sequences capable of binding to intrinsic membrane proteins (e.g., integrins) of cells, thereby obtaining cell-protein adhesion. In this specification, the terms “Arg-Gly-Asp” peptide or sequence or “RGD” peptide or sequence refer to a sequence containing at least one Arg-Gly-Asp capable of functioning as a binding site for an integrin receptor, and any peptide or amino acid sequence having a functional equivalent thereof.

[0204] The above microgel can be added to a suspension of mammalian (therapeutic) cells in sodium alginate or a similar gel-forming agent, and then gelled by dropping it into calcium chloride.

[0205] These may be added to cells deposited in other cell culture devices, including multi-well plates, to mitigate cell-cell interactions.

[0206] Vaccine use In another embodiment, we provide nano and microparticles modified to serve as delivery platforms for antigens in the context of vaccines. These nano and microparticles are: • It acts as a carrier for the antigen, such as an active but harmless virus including mRNA, RNA, DNA, proteins, viral shell fragments, a fully inactivated viral shell, or adenoviruses modified to express a desired antigenic protein. • Possesses a cationic group that can electrostatically bind antigens during storage and administration to the recipient's immune system. The adjuvant properties include being based on a cationic group, a polycationic group, or several carbohydrate groups, in order to ensure recognition and processing by the host immune system. The composition has cationic or polycationic groups that can be cleaved spontaneously or by enzyme-mediated hydrolysis to release an antigen payload that is bound over a time frame beneficial for inducing a strong immune response in the recipient. • The crosslinking agent can undergo slow spontaneous or enzyme-mediated hydrolysis to ensure final clearance, including renal clearance of microparticles from the recipient throughout the process. The composition comprises cationic and polycationic groups, as well as a non-stoichiometric amphoteric polymer electrolyte, which can bind antigens for storage at room temperature defined as up to 40°C, without requiring cold chain logistics during storage and transport. The cellular immune response can be enhanced by additional silver nanoparticles, either by co-precipitation during precipitation polymerization, by reductive precipitation from silver salts as part of post-functionalization, or by adsorption of pre-formed silver nanoparticles onto the described polymer nanoparticles.

[0207] Method for producing vaccine delivery particles Figure 4 shows two methods for forming nanoparticles for use as antigen carriers suitable for vaccine applications. Both methods involve initial precipitation polymerization 201, 206 of a transient crosslinking agent (e.g., methacrylic anhydride) and a gradually eroding divinyl crosslinking agent (e.g., disulfide crosslinked dimethacrylate) to ensure that the particles are ultimately removed by renal clearance. The second method involves the addition of a cationic monomer in the precipitation polymerization 206. The first method yields high-density as-formed microspheres 202, which can then be modified with diamines or triamines 203, or hydrolyzed or functionalized into a vinyl-functionalized polyanionic microgel 204, which is then grafted through with DMAEMA / DMAEA and anionic monomers to form a non-stoichiometric (cationic) amphoteric polyelectrolyte microgel 205. The second method yields microspheres 207 having cationic and anhydride crosslinks. These are then modified with diamines or triamines 208, followed by the formulation of an antigen and lyophilization 209.

[0208] As an example, submicron (0.1-0.9 micron) particles grafted with a suitable copolymer can be used as a vaccine delivery vehicle. Here, antigens based on proteins, mRNA, DNA, or viral or bacterial shell fragments or whole-inactivated viruses or bacteria or other pathogens may be absorbed into or bound to hydrogel particles post-functionalized with groups or polymers capable of binding to these antigens. This may include cationic modifying groups or non-stoichiometric amphoteric polymer electrolytes containing excess cationic charge in the grafted copolymer.

[0209] In addition to binding antigens, the cationic properties of the microgel can assist in the uptake of antigen-containing vaccine particles into macrophages or other cells during administration.

[0210] Similarly, the cationicity of the microgel surface can elicit an adjuvant response when introduced into tissues by injection, nasal administration, or other administration forms.

[0211] Alternatively or additionally, silver nanoparticles may be introduced into the vaccine particles to enhance adjuvant activity.

[0212] Similarly, cationic groups or copolymers can be designed to release antigens over a time frame suitable for inducing a sustained immune response by undergoing a charge shift to anionic groups.

[0213] Similarly, copolymers grafted to microgels can be designed to bind antigens in a way that prevents denaturation or other forms of inactivation during extended storage at high temperatures, including storage at -30 °C, -10 °C, +2 - 8 °C, or room temperature (defined as temperatures up to 37 °C or 40 °C). In particular, complex formation of native proteins with some synthetic or natural polymers can protect the proteins from denaturation. Examples include the native protein called the HERO protein described in 2020 10 and other intrinsically disordered proteins (IDPs). 11Similarly, synthetic amphoteric polyelectrolytes sequester native proteins and protect them from denaturation during heating or dry storage. Copolymers grafted to this microgel can be designed to form a coacervate phase that sequesters native proteins and prevents denaturation during dry storage after lyophilization (freeze-drying) using anionic microgels at physiological pH. Coacervates are defined as electrostatically maintained, highly hydrated polymer phases that contain a single polymer with near-stoichiometric balance of anionic and cationic charges (simple coacervates), or a pair or larger set of polymers and copolymers with cationic and anionic monomers in net stoichiometric or near-stoichiometric balance (complex coacervates). Alternatively, the microgel can form a complex coacervate phase after complexing with a grafted copolymer and other antigens such as proteins or RNA or DNA that are predominantly negatively charged, and can similarly protect the payload protein of the polynucleotide from denaturation or other degradation during storage. Finally, antigen-binding coacervates can be generated by electrostatically absorbing predominantly cationic polymers or copolymers onto anionic hydrolytic microgel particles.

[0214] In all of these cases, the effective charge of the coacervate phase should be neutral or preferably cationic to facilitate antigen uptake, cellular uptake of microgel particles, and adjuvant activity. Similarly, permanent crosslinkers can be designed to degrade over a time range suitable to allow for the eventual renal clearance of the injected vaccine particles.

Example

[0215] The following examples demonstrate the preparation of reactive particles based on MeAn, the conversion of reactive particles into various functionalized hydrogel particles, and some uses of the hydrogel particles. The preparation of reactive particles and their conversion to hydrogels are schematically shown in Figure 5.

[0216] All materials were used in the condition they were received, unless otherwise noted. 2,2'-Azobis(2-methylpropionitrile) (AIBN, 99.9%) was purchased from Dupont. Methacrylic anhydride (MeAn, 94%), ethylene glycol dimethacrylate (EGDMA, 98%), diethylene glycol dimethacrylate (DEGDMA, 95%), dimethoxypropane (DMPA, 98%), 2-hydroxyethyl methacrylate (HEMA, ≥99%), p-toluenesulfonic acid monohydrate (pTSA, ≥98.5%), 4-methoxyphenol (MEHQ, 99%), silica gel (technical grade, pore size 60 Å, 230-40 The following materials were purchased from Sigma Aldrich: 0 mesh, sand (50-70 mesh), potassium carbonate (anhydrous, 99%), 3-(dimethylamino)-1-propylamine (DMAPA, 99%), acetonitrile (ACN, ≥99.5%), methyl ethyl ketone (MEK, ≥99.0%), heptane (99%), acetone (≥99.5%), hexane (≥99.5%), ethyl acetate (≥99.5%), N,N-dimethylformamide (DMF, ≥99.8%), and chloroform-D (CDCl3, 99.8% D). Sodium chloride (NaCl, ACS reagent) and sodium hydroxide (NaOH, ACS reagent) were purchased from ACP Chemicals. Disodium hydrogen diphosphate heptahydrate (Na2HPO4·7H2O), sodium bicarbonate (NaHCO3), hydrochloric acid 35-37 wt% (HCl, reagent grade), glacial acetic acid (reagent grade), and sodium acetate (reagent grade) were purchased from Caledon Laboratories Ltd. Sodium dihydrogen orthophosphate (NaH2PO4·H2O, Assured grade) was purchased from BDH Chemicals. Deuterium oxide (D2O, 99.9% D) was purchased from Cambridge Isotope Laboratories Inc.Trypan blue 0.4%, phosphate-buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM, 4.5 g / L D-glucose, L-glutamine, 110 mg / L sodium pyruvate), 0.5% trypsin-EDTA (10X), penicillin-streptomycin, calf serum (BCS), and tetramethylrhodamine cadaverine, 5-(and-6)-((N-(5-aminopentyl)amino)carbonyl)tetramethylrhodamine (mixed isomers) (TAMRA cadaverine) were purchased from Thermo Fisher Scientific. 2-propanol (ACS certified) and dimethyl sulfoxide (DMSO, ≥99.7%) were purchased from Fisher Chemicals. RGD (Arg-Gly-Asp) (>95%) was purchased from Abcam.

[0217] The as-formed particles and / or particles after hydrolysis and / or functionalization are examined by microscopy. 1 The microgels were characterized by 1H NMR and zeta potential measurements. Bright-field images were acquired using a Nikon Eclipse LV100ND upright microscope or a Nikon Ti Eclipse inverted microscope. Confocal images were acquired using a Nikon A1 Confocal Ti Eclipse microscope. Microgel diameter was manually measured in bright-field images using two-point measurements with Nikon NIS-elements Advanced Research software. 1 ¹H NMR analysis was performed on particles swollen in D2O or DMSO-d6 using a Bruker 600 MHz analyzer. Zeta potentials were measured using a Malvern Zetasizer Nano ZS.

[0218] [Example 1] The following examples demonstrate the synthesis of MeAn-containing particles by thermally initiated and photoinitiated polymerization. To obtain microparticles, precipitation polymerization was performed according to the following scheme. The scheme shows the conditions for photoinitiated polymerization, but the same solution was used for thermally initiated polymerization heated at 60-70°C. In this example and the following examples, MeAn-based particles are represented as (MED-X / Y / Z), where X is the volume percentage of MEK in the MEK / heptane cosolvent mixture used, Y and Z are the molar percentages of the two permanent crosslinkers EGDMA and DEGDMA in the total monomer pool, and the remainder represents MeAn.

[0219] [ka]

[0220] Polymerization was typically carried out with a total monomer content of 5% (w / v) and 2 wt% AIBN relative to the total monomer. For example, MED-55 / 5 / 5 particles were prepared from MeAn (1.596 g, 10.3 mmol), EGDMA (0.114 g, 0.57 mmol), DEGDMA (0.139 g, 0.57 mmol), and AIBN (0.037 g) dissolved in 35.15 mL of a 55 / 45 (v / v) MEK / heptane mixture (19.33 mL MEK, 15.82 mL heptane). The mixed solvent was prepared by combining 15.56 g of MEK and 10.82 g of heptane by weight to produce a 55 / 45 mixture, using MEK with a density of 0.805 g / mL and heptane with a density of 0.684 g / mL at room temperature. Next, the reaction mixture was transferred to a 40 mL glass scintillation vial with a screw cap, and a PTFE septum was incorporated. For photoinitiated polymerization, the vial was placed on a series of steel rollers (VIVO Electric 12-roller hot dog and 5-roller grill cooker; model hotdg-v005) and rotated at 3.25 rpm for 5 hours at room temperature while being irradiated with an Everbeam 100 W 365 nm UV LED Black light set positioned 9 cm above the steel rollers. For thermal-initiated polymerization, the vial was similarly rotated along its long axis (4-8 rpm) in an oven (UVP HB-1000 Hybridizer, or similar) set to 70°C. After polymerization, the reaction mixture was transferred to a 50 mL centrifuge tube, and particles were isolated by centrifugation (4000 rpm, 3082 g, 15 min). The particles were purified by washing them three times with 40 mL of acetone, followed by one wash with 40 mL of ACN. The particles were then redispersed in a solvent and precipitated by centrifugation. After purification, the particles were redispersed in 40 mL of ACN or DMF for storage or functionalization. To measure the isolation yield, a fixed amount of 1 mL of a stirred ACN suspension of particles was transferred to a pre-weighed 20 mL scintillation vial, dried under nitrogen for 1 hour, and then the vial was placed in a desiccator and dried under vacuum at 20°C for 4 days. A microscopic image of MED-60 / 0 / 10 particles (thermal initiated) in DMF is shown in Figure 6. - Diameter: 1.74 ± 0.22 μm (CV 0.13).Similar MED-57 / 0 / 10 particles prepared using a 57:43 ratio of MEK / heptane (low polarity solvent) had a diameter of 2.74 ± 0.57 μm (CV 0.21).

[0221] MeAn can be an inefficient crosslinking agent because it has a strong tendency towards cyclic polymerization, and rearrangement of the anhydride group can lead to the loss of initially formed crosslinks. Furthermore, the conditions required for precipitation polymerization into microspheres (low total monomer content, slight solubility) tend to favor cyclic growth over acyclic growth of MeAn. However, the presence of comonomers reduced the degree of MeAn cyclic polymerization and thus increased MeAn crosslinking, as in this example where MeAn was paired with a permanent crosslinking agent. Figure 7 shows bright-field optical microscope images of MeAN-only (MED-55 / 0 / 0, optic) microspheres formed in 55 / 45 MEK / heptane in the absence of a permanent crosslinking agent. MED-55 / 0 / 0 microspheres were formed in isolation yield 38% (Table 1) and remained unchanged when dispersed in MEK or DMF. If they are not crosslinked and consist only of linear pMeAn chains, the solvent should cause dissolution or confluence of the particles. The successful formation of microspheres in reasonable yields demonstrated that MeAn acts as a crosslinking agent under these polymerization conditions.

[0222] When the permanent crosslinking agents EGDMA or DEGDMA, or a mixture thereof, were added at 10 mol% relative to the total monomer, particles were obtained under the same polymerization conditions. Particles prepared by photopolymerization in the presence of one or more permanent crosslinking agents were obtained with an isolated particle yield of approximately 40–55% (Table 1). The particle yield was higher with thermally initiated polymerization, at approximately 50–80% (Table 1).

[0223] [Table 1]

[0224] The MEK / heptane mixed solvent chosen for precipitation polymerization was advantageous because it was a small amount of solvent (low viscosity) that did not react with the anhydride. A further advantage of the MEK / heptane solvent system is that fine-tuning of the dissolving power is possible by changing the ratio of the two components.

[0225] Figures 8A-8F show that narrowly dispersed MED-55 / 5 / 5(light) particles can be produced with a monomer content of up to 7%, and that the size increases with the amount of monomer used. Sizes in the range of 1-3 μm or less were observed. Larger particles were seen at a 10% content, and a size of 5-6 μm could be obtained, but the size dispersion was poor. The size of all particles further increased after hydrolysis or functionalization.

[0226] Figure 9 shows that as the initiator concentration increases, the size of the MED-62 / 0 / 10(optical) particles gradually increases, resulting in the acquisition of narrowly dispersed particles. This is likely a result of higher monomer conversion.

[0227] Figure 10 shows the diameters of MED-X / 0 / 10(light) particles prepared in MEK / heptane containing 50–70% MEK. Varying the solvent polarity within this range has little effect on size. Samples prepared in solvents containing up to 62% MEK yielded particles with an average diameter of approximately 2 μm and narrow dispersion (CV ≤ 0.1). Particle size increases after hydrolysis or functionalization.

[0228] [Example 2] The anhydrous is hydrolyzed very rapidly in aqueous media, resulting in cleavage (crosslinking, cyclic, and suspension) of the anhydrous in the case of MeAn-based particles, and the generation of methacrylic acid or carboxylate groups depending on the pH. This causes the particles to swell at higher pH, or dissolve in the absence of permanent crosslinking agents, especially when the acidic groups are deprotonated. Hydrolysis of MeAn-based microspheres has been used to produce anionic microgels. For example, purified MED-55 / 5 / 5 (photo) microspheres were suspended in 40 mL of ACN, settled by centrifugation, resuspended in 5 mL of ACN, and then 11.3 mL of 1 M NaOH (1.1 equivalents) was added. After 30 minutes, the mixture was diluted to 40 mL with distilled water and then maintained overnight at room temperature under constant mixing at 20 rpm. The hydrolyzed microgel suspension was transferred to a cellulose dialysis tubing (3500 Da molecular weight cutoff (MWCO), Spectrum Laboratories) and purified by dialyzing against distilled water, changing the water daily, until the dialysate showed no absorbance by UV-Vis spectroscopy. The purified microgel was then freeze-dried to obtain a white solid. To prepare sterile microgels, the freeze-dried microgel was immersed in 70% ethanol for 2 hours, settled by centrifugation (3082 g, 15 minutes), re-swelled in sterile distilled water, and then freeze-dried under sterile conditions using a Labconco sterile adapter.

[0229] Microscopic images of hydrolyzed MED-60 / 0 / 10 (thermal) particles are shown in Figure 6. At pH 7.4 (Figure 6C), the particle diameter was 5.49–0.78 μm, and at pH 2, it was 1.6–0.4 μm, similar to the particle diameter before hydrolysis. The survival of the particles after hydrolysis indicates permanent crosslinking, and the dramatic swelling at pH 7.4 is consistent with the formation of a mildly crosslinked hydrogel. The DMSO-d6 suspension of acidified COOH-type MED-60 / 0 / 10 (thermal) particles... 1The \(^1H\) NMR analysis showed that the signals at 0.7 - 2.2 ppm (CH3, CH2 backbone), 3.5 - 4.2 ppm (CH2O), and 12.3 ppm (COOH) in a ratio of 76:8:12.5 were consistent with MAA:DEGDMA = 93:7. Since each MeAn molecule can give rise to two MAA units, this corresponds to MeAn:DEGDMA = 87:13.

[0230] MED - 55 / 0 / 0 (light) particles lacking a permanent cross - linker dissolved when the anhydride was hydrolyzed, while particles made with permanent cross - linkers such as MED - 55 / 10 / 0 or - 55 / 5 / 5 swelled and became more transparent but did not dissolve. It was extremely difficult or impossible to resolve particles made with a larger fraction of DEGDMA such as MED - 55 / 2 / 8 (light) or MED - 55 / 0 / 10 (light) by ordinary optical microscopy after hydrolysis, reflecting a very high degree of solvation and swelling. The aqueous solution of MED - 55 / 0 / 10 microgel could pass through a 0.45 μm pore syringe filter but could not pass through a 0.22 μm syringe filter, suggesting that although highly deformable, it was still particulate.

[0231] The effect of pH on the swelling of MeAn - based anionic microgels containing different permanent cross - linker compositions was determined by optical microscopy. Hydrolyzed MED - 55 / 10 / 0, MED - 55 / 5 / 5, and MED - 55 / 0 / 10 made by photopolymerization were dispersed at 0.05 wt% in 100 mM phosphate buffer (pH 2.4), 100 mM acetate buffer (pH 4.75), 100 mM phosphate buffer (pH 7.06), and 100 mM carbonate buffer (pH 10.0).

[0232] The swelling of hydrolyzed particles provides information about the degree of crosslinking and stiffness of the hydrogel. Crosslinked poly(methacrylic acid) (pMAA) particles, such as those formed by the hydrolysis of MED particles, disintegrate at low pH and swell highly at high pH when all MAA groups are ionized. Hydrolyzed MED particles, prepared with 10 mol% permanent crosslinker but with different EGDMA to DEGDMA ratios, were suspended in solution at pH 2.4, 4.75, and 7 (Figure 11), where the carboxylic acid groups should be fully protonated (neutral), half-ionized, and fully ionized, respectively. The particles clearly swelled as the pH increased, and they became more difficult to separate as their refractive index approached that of the solution.

[0233] The degree of swelling is determined by comparing the volume of the disintegrated particles at pH 2.4 with the volume at higher pH levels, and examining the relationship (D x / D 2.4 ) 3 [Here, D x D is the particle diameter at a given pH. 2.4 The diameter at pH 2.4 was estimated by using [the specified value]. As shown in Figure 12, the particles underwent considerable swelling (≧10×) at pH 7, indicating they were mildly crosslinked hydrogels. Swelling increased significantly by increasing the DEGDMA content in the permanent crosslinker. Greater swelling was consistent with longer, hydrophilic crosslinkers than EGDMA, although the magnitude of the change was unexpected. The results suggest that the size and swelling of microgels can be further tuned using crosslinkers with longer spacers, such as oligo / poly(ethylene glycol) methacrylate, and hydrophobic alkyl spacers, such as butanediol dimethacrylate.

[0234] [Example 3] Polymer-linked anhydride groups can also be used for post-polymerization modification via reaction with nucleophiles such as amines, alcohols, or thiols to induce the incorporation of modifiers, the formation of polymer-linked carboxylic acid groups, and the cleavage of anhydride crosslinks. Reactions of MeAn-based particles with one or more of the wide variety of available modifiers can yield particles with a broad range of properties. The hydrophobicity and charge of the particles can be altered, and groups can be introduced to impart various useful properties (e.g., fluorescence or radiolabeling, cell binding, drug release, etc.). Functionalization of MeAn-based particles allows for the preparation of particles that would normally be unattainable by direct precipitation polymerization of structurally similar monomer units, and, depending on the selected modifier and conditions, it is also possible to control whether the modification occurs throughout the particle or is largely confined to the particle surface. To demonstrate functionalization, particles such as those prepared in Example 1 were reacted with reagents that are fluorescent, cationic, and / or cell-binding motifs, as described below.

[0235] Amphoteric polymer electrolyte microgel Amphoteric polyelectrolyte microgels were prepared by functionalizing particles containing MeAn with excess DMAPA. For example, a 50:50 cationic:anionic amphoteric polyelectrolyte microgel was targeted by adding DMAPA (3.17 g, 31.1 mmol) to MED-55 / 5 / 5 (photo) microspheres suspended in 40 mL of ACN in a 50 mL centrifuge tube. This corresponds to approximately a 6-fold excess of DMAPA relative to the amount of MeAn in the particles. The reaction was maintained overnight at room temperature while mixing the vial / tube at 20 rpm. After the reaction, the microgel was settled by centrifugation (3082 g, 15 min), then washed once by resuspending in 40 mL of ACN, and then centrifuged again. After washing, the microgel was dispersed in 40 mL of distilled water, swollen, and then transferred to cellulose dialysis tubing (3500 Da MWCO). The microgel was first dialyzed twice over two days with 0.9 wt / v% NaCl, and then dialyzed again for four days with distilled water that was replaced daily. The microgel was then freeze-dried to obtain a white solid. It was then sterilized as described above.

[0236] Fluorescently labeled microgels As-formed MeAn-based microspheres were fluorescently labeled with TAMRA targeting a labeling degree of 0.025–0.05 mol% relative to the MeAn units. TAMRA-cadaverine (222 μL of 0.2 wt% DMF solution; 0.86 μmol) was added to a 30 mL ACN suspension of MeAn-based microspheres containing approximately 0.53 g (3.45 mmol) of polymer MeAn groups, and the mixture was then mixed at room temperature (22°C) for 2 days. The particles were isolated by centrifugation and then washed three times with 40 mL of ACN. The microspheres were then resuspended in 40 mL of distilled water, mixed at room temperature (22°C) for 1 day, isolated by centrifugation, resuspended in 10 mL of distilled water, and dialyzed with water using 3500 Da MWCO cellulose acetate tubing. The water bath was changed daily until the absorbance measurement of the dialysate reached 0, indicating no further elution of low molecular weight particles. The particles were isolated and resuspended in 40 mL of 70% (v / v) ethanol for 1 hour for sterilization. The ethanol suspension was centrifuged and then transferred to a biosafety cabinet, where the supernatant was removed. The particles were dispersed in 30 mL of sterile water, frozen with dry ice, and freeze-dried using a sterile adapter (Labconco) to obtain TAMRA-labeled particles as pink solids.

[0237] RGD-functionalized microgels MeAn-based microspheres were functionalized with both TAMRA and RGD to target functionalization levels (wrt MeAn) of approximately 0.025 and 0.5 mol%, respectively. A solution of RGD (5.3 mg, 15.3 μmol) in ACN / DMF (1:1 (v / v), 2 mL) was added to 30 mL of an ACN suspension of MeAn-based microspheres containing approximately 0.53 g (3.45 mmol) of MeAn units, and then, after approximately 10 minutes, TAMRA-cadaverine (222 μL of 0.2 wt% DMF solution; 0.86 μmol) was added. The reaction, washing, and isolation steps were carried out as previously described in this example. The lyophilized RGD and TAMRA-functionalized microspheres were isolated as pink solids.

[0238] Characteristics of functionalized particles DMAPA-functionalized MED-57 / 0 / 10 (thermal) particles in D2O 1 ¹H NMR analysis showed that the signals at 0.7–2.4 ppm (CH3, CH2 main chain), 2.85 ppm (N(CH3)2), 3.15 ppm (N-CH2), and 3.5–4.2 ppm (CH2O) with a ratio of 15.8:6:3.7:1.5 were consistent with MAA:DMAPMA (acid / amine) = 58:42. Particles resuspended in PBS at pH 2 and pH 7.4 were examined by microscopy. Particle diameters were 6.57 ± 1.01 μm at pH 7.4 and 7.06 ± 1.61 μm at pH 2. The large diameter of the precursor particles (DMF medium diameter: 2.74 ± 0.57 μm; CV 0.21) indicates that the transient crosslinking was cleaved, resulting in the formation of a lightly crosslinked hydrogel. In contrast to the hydrolyzed particles in Example 2, the particles did not disintegrate at low pH. This indicates that the functionalization was successful, as the presence of charged groups in the form of ammonium ions ensures that the particles remain swollen at low pH.

[0239] Figure 13A shows microscopic images of MED-60 / 0 / 10 (thermal) particles functionalized with N,N-dimethylethylenediamine (DMEDA) and then dispersed in HEPES buffered saline (pH 7.6). Figure 13B shows that the majority of the particles are 4-6 μm in size. 2 This is a plot of particle area showing that it has an area of ​​2.25 to 2.75 μm, corresponding to a particle diameter of 2.25 to 2.75 μm.

[0240] Images of MED-55 / 5 / 5(light) particles before and after functionalization with DMAPA and TAMRA-cadaverine (0.05 mol%) are shown in Figures 14A-C. The particles underwent dramatic swelling after functionalization and dispersion in aqueous solution and were able to form a densely packed array with a fairly uniform size (Figure 14B). Confocal fluorescence microscopy revealed that the TAMRA label was concentrated on the particle surface, presumably because TAMRA-cadaverine, added before DMAPA, reacted with the first MeAn group it encountered. This demonstrates the ease of particle functionalization, as well as the ability to localize different modifiers depending on the order of addition and / or molecular weight.

[0241] The reaction of MeAn groups in particles with diamines or polyamines allows for the production of particle charges tailored to specific biomaterial applications, starting from the same base scaffold particles. Hydrolysis of MeAn yields particles that are anionic at neutral pH, while the complete reaction of anhydrous groups with diamines such as DMAPA yields amphoteric polymer electrolyte particles with a charge ratio of approximately 1:1. MED-55 / 0 / 10(light) particles functionalized with excess DMAPA are suspended in D2O, 1 Analysis was performed by 1H NMR. They were found to have a degree of functionalization of approximately 80%, corresponding to a cationic:anionic charge ratio of 40:60, close to the target 50:50 ratio. The slightly excess anionic charge may be due to incomplete functionalization, the presence of some MAA in the MeAn starting material (94% purity), and / or accidental hydrolysis of some anhydride groups prior to functionalization.

[0242] MED-55 / 10 / 0 (photo) microspheres, either hydrolyzed only or functionalized with DMAPA, were dispersed in PBS (pH 7.4) at a concentration of 0.25 wt%. The zeta potential of each particle sample was measured using Malvern's Zetasizer Nano ZS. Approximately 700 μL of the particle dispersion was transferred to a Malvern Zeta-Dip Cell, and measurements (n=3) were performed at 25°C. Zeta potential measurements were performed to determine the surface charge of the hydrolyzed and DMAPA-functionalized MED-55 / 10 / 0 particles (Figure 15). Hydrolyzed MED-55 / 10 / 0 particles showed a strong negative zeta potential (-20.7 ± 4.7 mV) at physiological pH, as expected for lightly crosslinked pMAA particles. DMAPA-functionalized particles have a zeta potential close to zero (-1.82 ± 0.27 mV), which is consistent with amphoteric polymer electrolytes having a charge ratio of approximately 1:1.

[0243] [Example 4] In some biomaterial applications, it is desirable to incorporate controlled degradation of materials under physiological conditions. Acid-unstable functional groups such as acetals and ketals are of interest because they degrade under acidic conditions while maintaining stability under basic conditions. To demonstrate particulate degradation, a ketal-containing crosslinking agent (propane-2,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(2-methyl acrylate) (referred to here as KTMA) was prepared by an acid-catalyzed reaction of 2-hydroxyethyl methacrylate and dimethoxypropane. KTMA is structurally similar to DEGDMA and was expected to be usable under the same polymerization conditions.

[0244] KTMA was synthesized using a procedure based on previously reported synthesis methods. 12,13HEMA (10.0 g, 76.8 mmol), DMPA (3.805 g, 36.5 mmol), pTSA (0.157 g, 0.825 mmol), and MEHQ (0.20 g, 0.2% wrt HEMA) were placed in a 25 mL pear-shaped flask equipped with a magnetic stirring bar. The reaction mixture was heated overnight at 60°C in an oil bath while bubbling nitrogen gas through the mixture to remove methanol. The resulting dark red reaction mixture was cooled to room temperature and then passed through a 100 g silica stopper using an 80 / 20 hexane / ethyl acetate eluent. The fraction containing the product was collected and concentrated under vacuum to obtain KTMA as a pale yellow liquid in yield 21% with a purity of approximately 95%. 1 1H NMR (CDCl 3, The values ​​for 600MHz are shown in Figure 16: δ 6.09 (2H,s), 5.55 (2H,s), 4.26 (4H,t), 3.69 (4H,t), 1.93 (6H,s), 1.37 (6H,s).

[0245] The procedure described above (Example 1) was used, but KTMA-crosslinked MeAn microparticles were synthesized using a degradable KTMA crosslinking agent instead of EGDMA and / or DEGDMA. For example, MKT-55 / 15 particles (15 mol% KTMA, 55 / 45 MEK / heptane) were prepared by photoinitiated polymerization using MeAn (1.284 g, 8.3 mmol), KTMA (0.448 g, 1.49 mmol), and AIBN (0.036 g, 0.022 mmol) dissolved in 35.15 mL of 55 / 45 MEK / heptane. After isolation and washing, the supernatant was removed and the anhydride groups were hydrolyzed by adding a NaOH solution (20 mol excess relative to the MeAn units). After allowing the reaction mixture to stand for 1 day, the particles were isolated by centrifugation and resuspended in 40 mL of distilled water. The suspension had a pH of approximately 11, which is important to prevent immature degradation of the ketal groups.

[0246] Indeed, narrowly dispersed KTMA cross-linked spherical particles were obtained under the same conditions using EGDMA and DEGDMA, as evidenced by the formation of a densely packed particle array (Figure 17). After hydrolysis, particles prepared with 10 mol% KTMA (MKT-55 / 10) swelled significantly at physiological pH and decomposed quite rapidly. Increasing the KTMA content to 15 mol% (MKT-55 / 15) resulted in longer-lived particles.

[0247] Anionic MKT-55 / 15 microgels, prepared by selective hydrolysis of anhydride groups under basic conditions, were dispersed in buffer at pH 5, 7, and 10 at room temperature to explore the rate of particle degradation (Figure 18). At pH 5, particles visibly swelled within 15 minutes and disappeared after 30 minutes, while at pH 7, it took 75 minutes. At pH 10, the particles remained unchanged after 24 hours. The accelerated degradation rate at lower pH is consistent with the hydrolysis mechanism catalyzed by acids in the ketal and acetal. Slower degradation can be achieved by using higher KTMA content, introducing hydrophobicity via functionalization or copolymerization, or altering the properties of the ketal bonds.

[0248] [Example 5] The following examples demonstrate that polycations can be grafted onto hydrogel microparticles using residual vinyl groups derived from a permanent crosslinking agent.

[0249] Hydrolyzed particles from Example 2 were grafted with 2-(N,N-dimethylamino)ethyl acrylate (DMAEA). Hydrolyzed MeAn-DEGDMA(90:10) particles in acid (COOH) form (0.100 g) were combined with 10 mL DMF, 1.00 g DMAEA (7.00 mmol), 11.5 mg (1 mol%) AIBN, and optionally 28.0 mg (1 mol%) fluorescein O-methacrylate. The solution was bubbling with nitrogen for 45 minutes and then heated in an oil bath at 70°C for 18 hours. A certain amount of the reaction mixture 1¹H NMR (DMSO-d6) showed that the reaction using fluorescein O-methacrylate resulted in 58% monomer conversion, while the reaction without fluorescein O-methacrylate resulted in 83% monomer conversion. Once the reaction mixture cooled, the particles precipitated, and the yellow supernatant was removed. The particles were washed once by dispersing them in acetone (approximately 45 mL), then centrifuged, and then dispersed in 11 mL of approximately 0.1 M HCl. After adjusting the suspension to pH 2.2 using 0.1 M NaOH, it was transferred to a dialysis tubing (1 MDa cutoff), and dialyzed with 1 mM HCl (4 L) for 2 days, changing the bath once. The particles were isolated by lyophilization. 1 The 1H NMR spectrum includes signals at 0.7–2.2 ppm (CH3, CH2, CH main chain), 2.8 ppm (N(CH3)2), and 12.3 ppm (COOH) with a ratio of 26.7:6:3.89, consistent with MAA:DMAEA = 80:20.

[0250] [Example 6] cryopreservation This example demonstrates that the hydrogel microparticles prepared in the above examples can act as a cryoprotectant.

[0251] NIH 3T3 mouse fibroblasts were cultured in T-75 tissue culture-treated flasks in DMEM supplemented with 10% v / v BCS and 1% v / v penicillin-streptomycin, and the cells were maintained in an incubator at 37°C and 5% CO2. When the cells reached 70-90% confluence, they were washed with PBS and incubated with 0.05% trypsin-EDTA solution in PBS at 37°C for 2 minutes to detach the cells. The cells were quenched by adding supplemental DMEM to the trypsin-EDTA solution, collected, and transferred to a 15 mL centrifuge tube. The cells were centrifuged at 300 g for 5 minutes and resuspended in 5 mL of supplemental DMEM. A fixed volume of 50 μL of the resuspended cells was stained with 50 μL of 0.4% trypan blue, and cell viability and concentration were measured using an Invitrogen Countess automated cell counter. For cryopreservation, cells were prepared by transferring a fixed amount of resuspended cells to 15 mL centrifuge tubes using an appropriate cell suspension volume, achieving 4 million cells per tube. The cells were allowed to settle at 300 g for 5 minutes, resuspended in 1 mL of cryoprotective solution to achieve a cell concentration of 4 million cells / mL, and then transferred to 2 mL of polypropylene cryotubes. The cryotubes were placed in a Mr.Frosty container filled with isopropanol, and the container was then placed in a -80°C freezer to achieve a cooling rate of approximately 1°C / min. After 24 hours, the frozen samples were thawed in a 37°C water bath for 2 minutes, then diluted in 9 mL of preheated (37°C) DMEM and allowed to settle at 300 g for 5 minutes. The cells were resuspended in 1 mL of DMEM, and each sample was stained with 50 μL of 0.4% trypan blue to measure cell viability and concentration. To monitor cell adhesion and growth after thawing, the remaining portion of each cryopreserved sample was divided into three 300 μL portions and seeded into three wells of a 6-well tissue culture treatment plate containing 3 mL of DMEM per well. The plates were maintained and monitored in a 37°C, 5% CO2 incubator for 7 days after thawing. All samples that reached confluence during this period were detached with trypsin-EDTA and transferred from the 6-well plate to a T-75 flask.On days 3, 5, and 7 after thawing, cells from one well of each sample were washed with PBS and detached by incubation with 0.025% trypsin-EDTA at 37°C for 2 minutes. The detached cells were collected, transferred to 15 mL tubes, allowed to settle at 300 g for 5 minutes, resuspended in 1 mL of DMEM, stained with 50 μL of 0.4% trypan blue in 50 μL portions, and counted using a Countess automated cell counter.

[0252] NIH 3T3 cells were frozen using a 24-hour freeze / thaw cycle with standard mammalian cell freezing procedures, at concentrations of 10% and 5 wt / v% in MED-55 / 2 / 8(photo)DMAPA-functionalized amphoteric polyelectrolyte microgels and DMEM, along with a 10% v / v DMSO-positive control and a DMEM-only-negative control. Figure 19 shows the viability and percentage of recovered cells immediately after thawing, as measured by trypan blue staining.

[0253] Cells frozen with a 10 wt / v% microgel showed comparable post-thaw viability and recovery rates to those frozen with a 10% v / v DMSO sample, with the DMSO sample showing a slightly higher recovery rate (%). Both high cell viability and recovery rate (%) are important indicators of the effectiveness of cryoprotective agents. With linear amphoteric polyelectrolytes, cell death resulting from ice crystal formation that causes cell fragmentation may lead to an overestimation of their effectiveness as cryoprotective agents. Fragmented dead cells may not be detected, resulting in higher cell viability measurements but lower recovery rates (%). 14 Figure 19 also shows that efficacy increases as the microgel concentration increases from 5 wt / v% to 10 wt / v%. This trend is consistent with previous reports on linear amphoteric polyelectrolytes for cryopreservation and indicates improved intracellular dehydration during freezing, preventing intracellular ice crystal formation, reducing ice crystal size, and mitigating cell sedimentation during freezing. 14,15,16

[0254] In addition to measures taken immediately after thawing, thawed cells were seeded into tissue culture plates, and cell adhesion and growth were observed as long-term measures of cell health. As shown in Figure 20, cell adhesion and growth of samples frozen with 10 wt / v% microgel were similar to those of cells frozen in 10% v / v DMSO. Furthermore, bright-field images of adhered cells showed healthy 3T3 morphology after adhesion (Figure 21). Interestingly, this measure appears to indicate that amphoteric polyelectrolyte microgels have improved efficacy as cryoprotective agents compared to similar linear amphoteric polyelectrolytes. The mechanism of cryopreservation with linear amphoteric polyelectrolytes is not clear, but a key step is thought to be conformal coating of the polymer around the cells during freezing. 17 Incomplete removal of this coating from cells after thawing may result in poor adhesion and proliferation. While we do not wish to dwell on any particular theory, flexible amphoteric polymer electrolyte microgels may deform around cells, resulting in conformal coatings that are then more easily separated from cells by differential sedimentation, potentially leading to better cell adhesion and growth.

[0255] [Example 7] Protein (antigen) binding The following examples demonstrate that cationic microparticles, such as those prepared in Example 5, can bind to the antigen ovalbumin.

[0256] A 4 mL distilled aqueous solution of 1 mg / mL pDMAEA-grafted microparticles (prepared as described in Example 5) was added to approximately 100 μL of 1 wt% fluorescein-labeled ovalbumin (OVA-FITC) in phosphate-buffered saline (PBS) solution (pH 7.4). The solution was vigorously mixed for approximately 1 minute using a vortex mixer, and then the particles were isolated by centrifugation at 4000 rpm for 1 minute. The particles were resuspended in 1 mL of PBS and then examined by fluorescence microscopy. As shown in Figures 22A and 22B, the particles became fluorescent, clearly indicating that they had bound to OVA-FITC.

[0257] [Example 8] Microgel cell uptake This example demonstrates cell uptake of a microgel that has been appropriately functionalized (in this case with RGD, which is a cell adhesion motif) as prepared in Example 3.

[0258] NIH 3T3 mouse fibroblasts were cultured to 70-90% confluence, detached, and counted. After counting, the cells were allowed to settle in 300g for 5 minutes, then resuspended in an appropriate volume of DMEM, and measured in 2.0 × 10⁶ units. 6 Cell concentrations of cells / mL were achieved. To form microgel-cell complex clusters, three stock solutions (2 wt / v%) in DMEM were prepared from MED-55 / 5 / 5 (photo) microgels functionalized with a) TAMRA, b) TAMRA and RGD, and c) TAMRA and DMAPA. A certain amount of cell suspension, microgel stock solution, and DMEM were combined in wells of a Cellvis96 6-well glass-bottom plate to form 1.0 × 10⁶ cells. 6A series of 200 μL / well samples were prepared containing cells / mL and one of three microgel compositions at varying concentrations (0.01–1.0 wt / v%). Cells were incubated with the microgels at 37°C for 3 days to allow cell / microgel interactions before imaging. After incubation for 3 days, cells were stained with 50 μL of 10 μmol of calcein-AM in PBS solution and imaged using a Nikon A1 Confocal Ti Eclipse microscope.

[0259] Particle functionalization can be used to incorporate modifiers that promote cell binding or internal migration. MED-55 / 5 / 5 (optical) particles were modified with RGD to promote cell binding and with TAMRA to promote particle visibility (Example 3). NIH 3T3 cells were co-cultured for 3 days with three different types of TAMRA-labeled MED-55 / 5 / 5 particles: A) DMAPA-functionalized amphoteric polyelectrolyte, B) RGD-functionalized anionic, and C) anionic. After incubation, cells were stained with calcein-AM and imaged by confocal microscopy (Figures 23A-C). Cells incubated in the presence of a microgel functionalized with RGD, a tripeptide attachment motif, were found to undergo internal migration by the cells, as seen in Figure 23B, along with the appearance of the TAMRA-labeled microgel (red) within the cells. The particles also appeared to reject calcein staining, and when viewed through FITC channels, vacancies seemed to appear within the cells. Confocal images of 3T3 cells incubated with DMAPA-functionalized amphoteric polyelectrolyte particles (Figure 23A) or anionic particles (Figure 23C) show no signs of internal migration. This suggests that RGD uptake into microgels facilitates cellular uptake of the microgel and may be used for the delivery of payloads, such as antigens for vaccine drug delivery, to cells.

[0260] [Example 9] Cell scaffolds and co-encapsulation of cells and microgels A DMAPA-functionalized amphoteric polyelectrolyte MED-55 / 15 / 0 (photo) microgel, fluorescently labeled with 0.05% TAMRA cadaverine to the MeAn group, was prepared as described in Example 3. After hydrolysis, it was purified by centrifugation / resuspension three times in distilled water and twice in PBS, and then resuspended in 40 mL of PBS. NIH 3T3 mouse fibroblasts were cultured in a T75 cell culture flask as described above until 70-90% confluence, detached, counted, and 6.0 × 10⁶ cells were placed in PBS. 6 The cells were resuspended to an approximate cell concentration of cells / mL. To the resuspended cells, 34.7 μL of 1 mg / mL calcein AM solution was added to stain the cells with a calcein AM concentration of 4 μM. In a 96-well glass-bottom plate, 50 μL of stained cells, 50 μL of fluorescently labeled MED-55 / 15 / 0 (photo) anionic microgel, and 200 μL of PBS were mixed and imaged using a Nikon A1 Confocal Ti Eclipse microscope.

[0261] Hydrogel particles are of interest as cellular scaffolds because they can form 2D or 3D arrays, and the properties of the scaffold can be fine-tuned by mixing particles of different sizes, rigidity, or chemical properties. The easy functionalization of MeAn-based particles makes them ideal starting materials for the production of components in particle scaffolds. Figure 24 shows a simple illustration of a 3D particle scaffold in which NIH 3T3 cells (stained with calcein-AM, green) were dispersed in a TAMRA-labeled amphoteric polyelectrolyte MED-55 / 15 / 0 (light) microgel.

[0262] The ease of handling particles means they can be easily combined with cells in more enclosed geometric shapes such as capsules. Calcium alginate is often used to encapsulate cells, but it can sometimes provide an environment that is not ideal for cells in terms of viability, differentiation, or proliferation. Co-encapsulation with particles that have suitable binding or signaling motifs for cells can provide an improved environment. As an example, NIH 3T3 cells were co-encapsulated in calcium alginate capsules with MED-55 / 10 / 0 (photo) amphoteric polyelectrolyte microgels at various concentrations (0.001–0.5%). Subsequently, the capsules were coated with a protective polycation / polyanion, and the encapsulated cells were stained live / dead with calcein-AM and ethidium-homodimer.

[0263] NIH 3T3 mouse fibroblasts were co-encapsulated in calcium-alginate capsules with MED-55 / 10 / 0 (photo) anionic microgels. 4 × 10 6 A 3T3 cell solution at a concentration of cells / mL was prepared in pH 7.4 35mM HEPES-buffered saline and combined in various ratios with 2 wt / v% Na-alginate solution in pH 7.4 35mM HEPES-buffered saline, 2 wt / v% MED-55 / 10 / 0 anionic microgel solution in pH 7.4 35mM HEPES-buffered saline, and pH 7.4 35mM HEPES-buffered saline, each totaling 1 mL, with a constant cell and Na-alginate concentration of 2 × 10⁶. 6Solutions containing microgels at concentrations of 0.5, 0.05, and 0.001 wt / v% were prepared using cells (s / mL) and 1.0 wt / v% Na-alginate. The prepared solutions were placed in three 1 mL BD plastic syringes and extruded through a Rame-Hart 20G coaxial needle at a solution flow rate of 15 mL / hour and coaxial airflow rate of 2.25 L / min, controlled by a Harvard Apparatus syringe pump, to form capsules. These capsules were then sheared into a gelling bath of 100 mM CaCl2, 45 mM NaCl, and 35 mM HEPES (pH 7.6). After extrusion, the formed capsules were gelled in the gelling bath for 5 minutes. The formed capsules were collected, washed, and then coated with poly-L-lysine (PLL) and partially (50%) hydrolyzed poly(methyl vinyl ether-alt-maleic anhydride) (PM50) to form capsules with a covalent crosslinked shell. The coated capsules were transferred to a 60 mm petri dish containing 5 mL of DMEM supplemented with 10% v / v BCS and 1 v / v% penicillin-streptomycin, and maintained in an incubator at 37°C and 5% CO2. After incubation for 1 day, approximately 200 μL each of capsules prepared at three concentrations were transferred to a 96-well glass-bottom plate, stained for 30 minutes with 50 μL of 10 μm and 50 μL of 4 μm ethidium homodimer solutions prepared in 35 mM HEPES-buffered saline, and then imaged using a Nikon A1 Confocal Ti Eclipse microscope.

[0264] As shown in Figures 25A-B, the particles did not interfere with the encapsulation process, and the encapsulated cells showed high viability in the presence of the particles. The capsule shown in Figure 25A exhibited the highest particle content, which affected the transparency of the capsule. Figure 25B shows 3T3 cells in close contact with particles inside the capsule. Cells can adhere to and diffuse to these amphoteric polyelectrolyte particles, but adhesion and diffusion are facilitated by the inclusion of cell-binding motifs that allow the particles to serve as granular cell attachment sites. References:

[0265] Table 2

Claims

1. The steps include combining at least one temporary crosslinking agent and at least one permanent crosslinking agent in an organic solvent suitable for precipitation polymerization, The steps include: causing the aforementioned precipitation polymerization to occur, thereby forming microparticles having a polymer containing monomers of the temporary crosslinking agent and the permanent crosslinking agent; Includes, The solvent has a Hildebrand solubility parameter that is 4 to 5 MPa 1 / 2 higher or lower than the Hildebrand solubility parameter of the polymer. The aforementioned temporary crosslinking agent is formula (I) or (IIa) to (IIf): 【Chemistry 1】 (In the formula, R1 and R2 are independently selected from H, a C1-C4 linear or branched carbon chain, benzyl, phenyl, or OJ, and J is defined as a C1-C4 linear or branched carbon chain.) 【Chemistry 2】 (In the formula, n is an integer between 1 and 3.) 【Transformation 3】 (In the formula, R3 is independently H or methyl.) It is a crosslinking agent, The permanent crosslinking agent is selected from the group consisting of divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), N,N'-methylenebisacrylamide (MBA), oligo / polyethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 1,6-hexanediol dimethacrylate. Methods for generating microparticles.

2. The method according to claim 1, wherein the total amount of monomers before precipitation polymerization is calculated as a combined amount of at least one temporary crosslinking agent, at least one permanent crosslinking agent, and any other monomers, and has a value of 1 to 20% by weight.

3. The method according to claim 1, wherein the total amount of crosslinking agent before precipitation polymerization is equal to the combined amount of the temporary crosslinking agent and the permanent crosslinking agent, and is greater than 10 mol%, and the ratio of the temporary crosslinking agent to the permanent crosslinking agent is 50:50 to 99:1 mol%.

4. The method according to claim 1, wherein the solvent is selected from the group consisting of acetonitrile, methyl ethyl ketone, heptane, and a combination of methyl ethyl ketone and heptane.

5. The method according to claim 1, wherein the temporary crosslinking agent is methacrylic anhydride or acrylic anhydride.

6. The method according to claim 1, wherein the permanent crosslinking agent has two or more vinyl groups.

7. The method according to claim 1, wherein the permanent crosslinking agent is 1 to 30 mol% of the total amount of monomers.

8. The method according to claim 1, wherein the combining step includes the step of combining photoinitiators, and the method further includes the step of irradiating the photoinitiators.

9. The method according to claim 1, wherein the precipitation polymerization is carried out without the addition of a surfactant and / or stabilizer, and / or the microparticles contain less than 0.1% of a surfactant and / or stabilizer.

10. The method according to claim 1, further comprising the step of functionalizing the monomer of the temporary crosslinking agent.

11. The method according to claim 10, wherein the functionalization step includes a step of functionalizing so as to obtain amine and carboxylic acid units in a ratio of 3:1 to 1:

3.

12. The method according to claim 11, wherein the ratio is 2:1 to 1:

2.

13. Microparticles comprising at least one polymer, wherein the at least one polymer comprises a polymer backbone containing a temporary crosslinking agent and a permanent crosslinking agent. The aforementioned temporary crosslinking agent is of formula (IIIa), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIi), (IIIj), (IIIk), (IIIl), (IIIm), (IIIin), and / or (IIIo): 【Chemistry 4】 (In the formula, R １ and R ２ H, C １ ~C ４ A linear or branched carbon chain, benzyl, phenyl, or OJ can be independently selected, where J is C １ ~C ４ Linear or branched carbon chains, 【Transformation 5】 (In the formula, n is an integer between 1 and 3.) 【Transformation 6】 (In the formula, R3 is independently H or methyl.) It is defined as, The permanent crosslinking agent is selected from the group consisting of divinylbenzene (DVB), ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA), N,N'-methylenebisacrylamide (MBA), oligo / polyethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 1,6-hexanediol dimethacrylate. Microparticles that are narrowly dispersed or monodisperse, have a size distribution with a coefficient of variation of less than 0.3, and do not contain surfactants and / or stabilizers.

14. A step of preparing microparticles obtained by the method of claim 1, The steps of functionalizing the aforementioned microparticles, The steps include bringing the cells into contact with the microparticles, The steps of freezing the cells and A method for cryopreserving cells, including...

15. A step of preparing microparticles obtained by the method of claim 1, The steps include functionalizing the aforementioned microparticles so that they act as carriers for an antigen, The steps of linking the antigen to the carrier and A method for generating a vaccine delivery platform, including [the following].

16. A step of preparing microparticles obtained by the method of claim 1, The steps of functionalizing the aforementioned microparticles, The steps include combining functionalized microparticles with cell and capsule-forming materials, The steps include: gelling the capsule-forming material so that the particles and cells are trapped inside the capsule; A method for producing encapsulated cells, including [the specified element].

17. The method according to claim 16, wherein the capsule-forming material is alginate.

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