Hydrogel Materials

Abstract

The present invention relates to a hydrogel material comprising a first hydrogel containing 0.15 to 21 wt % of a functionalized triblock molecule having the following formula (1): [Formula 1] TIFF2023543994000011.tif26161(1) Here, n is 4 to 680, and m is 1 to 10, based on the total weight of the first hydrogel. The first hydrogel contains 0.85 to 3.3 wt % silica based on the total weight of the first hydrogel and 75 to 99 wt % aqueous liquid based on the total weight of the first hydrogel, and the -Si-OH groups of the silica form -Si-O-Si- bonds with the -Si-(O)3- groups of the functionalized triblock molecule of formula (1).

Description

[Technical Field]

[0001] The present disclosure relates to hydrogel materials that are particularly useful for the controlled delivery of biologically active agents. [Background technology]

[0002] Hydrogels typically contain a network of polymer chains or aggregated colloids as the continuous solid phase and water as the dispersed liquid phase. The three-dimensional solid results from hydrophilic polymer chains held together by crosslinks or from colloids, e.g., formed from aggregated colloidal particles. Hydrogels are natural or synthetic polymer networks in which the liquid phase is water. They can be used as scaffolds for tissue engineering, as carriers for drugs or cells, or as drug delivery systems for sustained release of drugs.

[0003] In this specification, the following terms are used with the following meanings:

[0004] A sol is a fluid, homogeneous mixture, i.e., a colloidal dispersion, of at least one liquid phase and one solid phase, where the liquid phase is the continuous phase, e.g., water, ethanol, precursor residues, etc., and the solid phase is in the form of colloidal particles or polymers, e.g., triblock molecules, functionalized triblocks, and silica, as used herein, that are uniformly dispersed in the liquid phase.

[0005] A gel is a non-flowable homogeneous mixture of at least one solid phase and one liquid phase, i.e., a colloidal dispersion, in which the solid phase, such as the triblock molecule, functionalized triblock, and silica, forms a continuous solid phase, and the liquid, such as water, ethanol, or residues of a solid phase precursor, is uniformly dispersed throughout the continuous solid phase. The solid phase is typically an aggregated or cross-linked molecular structure formed by a chemical reaction (eg, by polymerization such as polycondensation) or by aggregation of colloids such as nanoscale particles. In a gel, a solid phase that is aggregated and / or crosslinked forms a continuous network throughout a defined space, and a liquid phase is uniformly dispersed throughout the continuous solid network.

[0006] The sol-gel transition is a term that refers to the process by which a sol changes to a gel. The sol-gel transition typically occurs when colloidal particles and / or polymers aggregate, the aggregates increase in size, and ultimately the sol transforms into a gel, where the aggregates form a continuous solid phase throughout the volume of the system without phase separation of the liquid phase, i.e., the liquid phase remains uniformly dispersed in the continuous solid phase.

[0007] A hydrogel is a water-based gel in which the liquid phase is water or contains more than 50% water by weight. A continuous hydrogel is a hydrogel in a single three-dimensional structure. A dispersed hydrogel is a set of hydrogel particles formed by breaking down a continuous three-dimensional hydrogel into smaller hydrogel particles, typically mixed with another continuous hydrogel.

[0008] In the context of the present invention, a flowable mixture refers to a material, for example a sol, that has viscoelastic properties due to a solid and a liquid phase, but in which the flowable liquid phase predominates over the solid phase. In the context of the present invention, a non-flowing mixture refers to a viscoelastic material, such as a hydrogel, which does not have flow properties at rest because the solid phase predominates over the liquid phase. In the case of flowing mixtures, the viscous properties (viscosity / loss modulus denoted G'' as determined by rheological methods) dominate over the elastic properties (elastic modulus / storage modulus denoted G'). Similarly, in a non-flowing mixture, the elastic properties (G') dominate over the viscous properties (G''). G' and G'' may be measured in the linear viscoelastic region under small angle oscillatory shear, for example by oscillatory measurements using a rheometer with a cone-plate or plate-plate geometry.

[0009] In the context of the present invention, a hybrid material is a material that comprises two or more components, such as, for example, a triblock molecule and silica, where there is a chemical bond between the components. Furthermore, when the term hybrid is used herein, it refers to a solid phase that includes at least two components that together form a solid phase in a hydrogel.

[0010] Vol-% represents volume percent and wt-% represents weight percent.

[0011] Nanoparticles (typically 1-100 nm in size) and colloids (typically 1-1000 nm in size) have overlapping ranges of values ​​in the 1-100 nm range. However, they have different chemical and physical properties. Nanoparticles typically have different material properties by larger amounts compared to the same material (i.e., material with the exact same chemical composition). For example, surface properties may be different, which can lead to different properties in terms of hydrophobicity and electrical conductivity. Colloids between 100-1000 nm in size begin to lose the special chemical properties of nanoparticles because gravity is still weaker than Brownian motion. Therefore, Brownian motion plays a major role in the production of various dispersions such as gels, suspensions, emulsions, and foams, where the goal is to obtain stable dispersions. Lyophobic colloid is the term used for colloidal dispersions where the colloid is not thermodynamically stable (although it may be stable for 15 or even 100 years). Lyophilic colloids are thermodynamically stable but are rarely used.

[0012] Nanoparticles or lyophobic colloids generally refer to certain molecular clusters that have reached a size and chemical structure that makes them practically insoluble in the surrounding liquid. However, nanoparticles or colloids are so small in size that they remain uniformly dispersed in the surrounding liquid, even when their density is higher than that of the liquid. This is because Brownian motion is stronger than gravity. Therefore, as nanoparticles and colloids move through a liquid, they can collide with each other and form larger structures. For example, they can aggregate, and sometimes they can even polymerize. Thus, colloidal particles are aggregates formed through chemical bonds and weaker interactions (such as van der Waals interactions), whereas the particles herein are formed when a three-dimensional gel is mechanically broken down to form particles, and thus include colloidal particles aggregated therein.

[0013] The article "Radiopaque Organic-Inorganic Hybrids Based on Poly(D,L-lactide)", Mazzocchetti et al., Biomacromolecules 2007, 8, 672-678, discloses hybrid organic-inorganic nanocomposites prepared from α,ω-triethoxysilane-terminated poly(D,L-lactic acid), which can be used as potential radiopaque biocompatible coatings for medical devices. The components of the composite and the composite itself are not all water soluble. Furthermore, in this publication, tetraethyl orthosilicate (TEOS) is added directly to the system. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Radiopaque Organic-Inorganic Hybrids Based on Poly(D,L-lactide)”, Mazzocchetti et al., Biomacromolecules 2007, 8, 672-678 Summary of the Invention [Problem to be solved by the invention]

[0015] (Objectives and Overview) The object of the present invention is to provide hydrogels that are suitable for the controlled delivery of biologically active agents, and are particularly useful for the protective encapsulation of various biologically active agents. One particular goal is encapsulation to ensure thermal stability of the biologically active agent. Another specific goal is to provide a hydrogel material that is injectable yet capable of encapsulating biologically active agents. [Means for solving the problem]

[0016] The present specification relates to a hydrogel material as follows. A hydrogel material comprising a first hydrogel, the first hydrogel comprising: 0.15% to 21% by weight of a functionalized triblock molecule having a structure of the following formula (1): [ka] (1) Here, n is 4 to 680 and m is 1 to 10 based on the total weight of the first hydrogel. - 0.85 to 4.0 wt. % silica, based on the total weight of the first hydrogel; and - 75 to 99 wt. % of an aqueous liquid based on the total weight of the first hydrogel; A hydrogel material, wherein the -Si-OH groups of the silica form -Si-O-Si- bonds with the -Si-(O)3- groups of the functionalized triblock molecule of formula (1).

[0017] This specification also relates to the use of hydrogel materials for the controlled delivery of biologically active agents and for the protective encapsulation of biologically active agents. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows a schematic representation of the structure of a functionalized triblock molecule. [Figure 2]FIG. 2 shows a schematic diagram of silica nanoparticle aggregates in a silica sol. [Figure 3] FIG. 3 shows a schematic structure of a reaction product between silica and a functionalized triblock molecule, according to one embodiment. [Figure 4] FIG. 4 shows the structure of a hydrogel material according to one embodiment. [Figure 5A] FIG. 5A shows the structure of a hydrogel material according to another embodiment. [Figure 5B] FIG. 5B shows the structure of a hydrogel material according to another embodiment. [Figure 6A] Figure 6A shows the cumulative release of antigen (norovirus P-particles) for the continuous hybrid / composite hydrogel R217-02 and the two-phase hybrid / composite hydrogels #1 (R217-0.2 / R40-0.2A and R217-0.2 / R40-0.2B) at 4 and 330 days after storage at room temperature (25 °C). [Figure 6B] FIG. 6B shows the cumulative release of silica. [Figure 7] FIG. 7 shows the cumulative release of antigen (norovirus P-particles) and silica dissolution of two-phase hybrid / composite hydrogel #2C. [Figure 8] FIG. 8 shows the cumulative release of antigen (norovirus P-particles) and silica dissolution of two-phase hybrid / composite hydrogel #2D. [Figure 9] FIG. 9 shows the cumulative release of eGFP and silica dissolution for the sequential hybrid / complex R217-0.2 with 10 μg of eGFP in 100 μl of hydrogel. [Figure 10] FIG. 10 shows the cumulative release of eGFP and dissolution of silica for the sequential hybrid / complex R217-0.2 with 20 μg of eGFP in 100 μl of hydrogel. [Figure 11] FIG. 11 shows the cumulative diffusion of eGFP from the sequential hybrid / complex R217-0.2 with eGFP at a hydrogel concentration of 10 μg / 100 μl. [Figure 12]FIG. 12 shows the cumulative diffusion of eGFP from the sequential hybrid / complex R217-0.2 with eGFP at a hydrogel concentration of 20 μg / 100 μl. [Figure 13] Figure 13 shows the extinction coefficients (G'' / G') for the continuous hybrid / composite hydrogel R217-0.2 and for the two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) at 4 and 28 days after storage in a syringe placed in an aluminum foil bag at room temperature (25 °C). [Figure 14] Figure 14 shows the damping coefficient (G'' / G') for the continuous hybrid / composite hydrogel R217-0.2 and for the two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) after 360 days of storage at room temperature (25°C). [Figure 15] FIG. 15 shows the dynamic viscosity for the continuous hybrid / composite hydrogel R217-0.2 and for the two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) after 4 days of storage at room temperature (25° C.). [Figure 16] FIG. 16 shows the dynamic viscosity for the continuous hybrid / composite hydrogel R217-0.2 and for the two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) after 1 day of storage in sealed aluminum foil bags at room temperature. [Figure 17A] FIG. 17A shows the thermal stability of norovirus P particles as a fraction of 15 nm particles at different temperatures as measured by dynamic light scattering (DLS). [Figure 17B] FIG. 17B shows the difference in the volume of distribution for P-particles measured at 50°C and 55°C. [Figure 18A] FIG. 18A shows the total volume of 15 nm aggregates of norovirus P-particles released from continuous hybrid / composite hydrogel R217-0.2 held in solution at the indicated temperatures as determined by DLS. [Figure 18B]FIG. 18B shows the total volume of 15 nm aggregates of norovirus P-particles released from controlled particles held in solution at the indicated temperatures as determined by DLS. [Figure 19] FIG. 19 shows a TEM image of norovirus P-particles dissolved from the hydrogel. [Figure 20A] FIG. 20A shows the kinetics of serum IgG antibodies in mice after immunization with two doses of 10 μg of norovirus P-particles (pp) alone or formulated with sequential hybrid / composite R217-0.2 hydrogel. [Figure 20B] Figure 20B shows the kinetics of serum IgG antibodies in mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 hydrogel. [Figure 21A] FIG. 21A shows the endpoint titers of serum IgG in mice after immunization with two doses of 10 μg of norovirus P-particles (pp) alone or formulated in sequential hybrid / complex R217-0.2 hydrogel. [Figure 21B] Figure 21B shows the endpoint titers of serum IgG in mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 / R40-0.2 hydrogel. [Figure 22A] FIG. 22A shows serum IgG1 endpoint titers in mice after immunization with two doses of 10 μg norovirus P-particles (pp) alone or formulated in sequential hybrid / composite R217-0.2 hydrogel. [Figure 22B] Figure 22B shows serum IgG1 endpoint titers in mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 / R40-0.2 hydrogel. [Figure 23A]FIG. 23A shows the endpoint titers of serum IgG2a in mice after immunization with two doses of 10 μg of norovirus P-particles (pp) alone or formulated in sequential hybrid / composite R217-0.2 hydrogel. [Figure 23B] Figure 23B shows serum IgG2 endpoint titers in mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 / R40-0.2 hydrogel. [Figure 24A] FIG. 24A shows the avidity of serum IgG antibodies in mice after immunization with two doses of 10 μg of norovirus P-particles (pp) alone or formulated in sequential hybrid / composite R217-0.2 hydrogel. [Figure 24B] Figure 24B shows the avidity of serum IgG antibodies in mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 / R40-0.2 hydrogel. [Figure 25A] Figure 25A shows cross-reactive serum IgG responses to heterologous NoV VLPs in mice after immunization with two doses of 10 μg of norovirus P-particles (pp) alone or formulated in sequential hybrid / composite R217-0.2 hydrogel. [Figure 25B] Figure 25B shows cross-reactive serum IgG responses to heterologous NoV VLPs in mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 / R40-0.2 hydrogel. [Figure 26A] Figure 26A shows the homogenous blocking of GII.4 VLP binding to the HBGA receptor by serum antibodies from mice after immunization with two doses of 10 μg of norovirus P-particles (pp) alone or formulated in sequential hybrid / composite R217-0.2 hydrogel. [Figure 26B]Figure 26B shows the homogenous blocking of GII.4 VLP binding to the HBGA receptor by serum antibodies from mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 / R40-0.2 hydrogel. [Figure 27A] Figure 27A shows the endpoint titers of fecal IgG antibodies in mice after immunization with two doses of 10 μg of norovirus P-particles (pp) alone or formulated in sequential hybrid / composite R217-0.2 hydrogel. [Figure 27B] Figure 27B shows the endpoint titers of fecal IgG antibodies in mice after immunization with a single dose of 20 μg of norovirus P-particles (pp) alone or formulated in two-phase hybrid / complex #1 R217-0.2 / R40-0.2 hydrogel. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present specification relates to a hydrogel material comprising a first hydrogel, the first hydrogel comprising: - 0.15% to 21% by weight of a functionalized triblock molecule having a structure of formula (1): [ka] (1) Here, n is 4 to 680 and m is 1 to 10 based on the total weight of the first hydrogel. - 0.85 to 4.0 wt. % silica, based on the total weight of the first hydrogel; and - 75 to 99 wt. % of an aqueous liquid based on the total weight of the first hydrogel; The -Si-OH groups of the silica form -Si-O-Si- bonds with the -Si-(O)3- groups of the functionalized triblock molecule of formula (1).

[0020] The present hydrogel material has several useful properties in a variety of fields. It is known to be highly suitable for the controlled delivery of biologically active agents. Some particularly suitable biologically active agents that can be used with the present hydrogel material are vaccine antigens and active pharmaceutical ingredients. The hydrogel material can also be made into an injectable form to facilitate its administration. Therefore, the rheological properties of the hydrogel material can be adjusted over a wide range, potentially allowing for tailoring of its injectability. By injectable, we mean a product that can be injected through a fine needle, such as a 25-27G needle, or a 23-30G needle, most typically a 25-30G needle. The hydrogel is non-flowable at rest, e.g., during storage, regardless of the amount of aqueous liquid in it. However, when the amount of aqueous liquid is sufficiently high, e.g., 96-99 wt %, the hydrogel flows through a needle and becomes injectable when pressure is applied, e.g., by a syringe plunger. Thus, such hydrogels are shear-thinning.

[0021] The material and all of its components are water-soluble. The liquid in the hydrogel is aqueous, which is particularly important for the administration of immunomodulatory and therapeutically active agents. Indeed, such uses require that the biological activity of the drug be maintained not only during the manufacture of the end product, but also in the final product. Furthermore, during drug delivery in the body, drug release is primarily controlled by the biological degradation of the hydrogel material. This biological degradation occurs primarily through dissolution into the aqueous phase of tissue fluids. All components of the hydrogel dissolve in the aqueous phase of tissue fluids. The polymer also degrades through enzymatic reactions, and enzymes are active in the aqueous phase. A particularly interesting advantage is that encapsulating biologically active agents within the present hydrogel materials ensures their thermal stability as a function of storage time and temperature, i.e., their biological activity can be maintained for extended periods of time. The hydrogel material also allows for a constant release rate of the biologically active agent upon administration.

[0022] The functionalized triblock molecule is sometimes called a hybrid, oligomer, or polymer. It is end-capped with silica. The -Si-O-Si- bond between the -Si-OH group of silica and the -Si-(O)3- group of the functionalized triblock molecule of formula (1) is a chemical bond. As used herein, the materials are also generally referred to as hybrid / composite, as some of the materials are hybrids and others are in composite form.

[0023] The hydrogel material can be one continuous hydrogel or can be composed of dispersed hydrogel particles uniformly dispersed within a continuous hydrogel.

[0024] In the above formula (1), n ​​is 4 to 680, and m is 1 to 10. The value of n (depending on the value of m) can be, for example, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 120, 135, 150, 200, 225, 270, 300, 350, 400, 450, 500, 550 or From 600 to 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 120, 135, 150, 200, 225, 270, 300, 350, 400, 450, 500, 550, 600 or 680. The value of m is, for example, from 1, 2, 3, 4, 5, 6, 7 or 8 up to 2, 3, 4, 5, 6, 7, 8, 9 or 10 (depending on the value of n).

[0025] Therefore, the hydrogel material has various forms summarized in Table 1 below. The type names shown in Table 1 are used herein.

[0026] The various types of hydrogel materials are described in more detail below.

[0027] [Table 1]

[0028] According to one embodiment, the functionalized triblock molecule of formula (1) and silica form colloidal particles, the network of said particles forming a continuous solid phase within which an aqueous liquid phase is uniformly distributed, said solid phase and aqueous liquid phase being in a single hydrogel entity, which is a continuous hybrid / composite of Table 1.

[0029] In this embodiment, the hydrogel is thus a homogeneous mixture of solid particle aggregates (colloidal aggregates formed from colloidal particles) and an aqueous liquid. Silica, at least partially in the form of colloidal particles and colloidal aggregates, forms chemical bonds with the triblock molecules to create a hybrid material. Colloidal silica particles are formed from spherical clusters of molecules, and their surface atoms can form chemical bonds with other compounds within the material, including not only the triblock molecules but also other colloidal silica particles.

[0030] Both the functionalized triblock molecules and silica are nanoscale structures: the functionalized triblock molecules are soluble or partially soluble in aqueous liquids, and the silica is in the form of nanoparticles. Hybrids / composites are formed when the Si-OH- groups of silica form -Si-O-Si- bonds with the Si-O- groups at the ends of the functionalized triblock molecules. One functionalized triblock molecule can also use these bonds to link with another functionalized triblock molecule, thus forming a network. Typically, each -Si-(O)3- group may bond to 0 to 3 functionalized triblock molecules or 0 to 3 silica molecules.

[0031] Additionally, silica nanoparticles can also bond (or aggregate or agglomerate through weak interactions) to one another. The silica nanoparticles may also form larger aggregates, but at least a portion of the nanoparticles are bound to at least one functionalized triblock molecule.

[0032] The silica in these hydrogel materials is in the form of colloids, nanoparticles, or aggregated nanoparticles (which are also colloids). Four oxygen atoms in silica are bonded to other molecules, such as other silicon atoms. Silica tends to form ring structures, and ring (i.e., spherical) silica molecules grow very quickly to such a size that they become insoluble in the surrounding liquid, hence the name nanoparticles or colloids. It does form its own solid phase in the surrounding liquid, but it is too small to remain stable in the liquid and to move around by Brownian motion in the same way as soluble molecules. There are many free Si-OH- groups on the surface of the particles, which can react with the functionalized triblock molecules to form chemical bonds and form hybrids / composites.

[0033] In this first type of material, the nanostructures—functionalized triblock molecules and silica nanoparticles—aggregate (or polymerize) and, if aggregation proceeds sufficiently, form a non-flowable hydrogel with the aqueous liquid. If the hydrogel material is used to encapsulate another molecule, this molecule is added to the mixture before the non-flowable hydrogel is formed.

[0034] According to another embodiment, the hydrogel material further comprises a second hydrogel, the second hydrogel comprising: - 0.15 to 0.8 wt. % of the functionalized triblock molecule of formula (1), based on the total weight of the second hydrogel; - 0.85 to 3.3 wt. % silica based on the total weight of the second hydrogel; and - 75 to 99 wt % of an aqueous liquid based on the total weight of the second hydrogel, wherein the -Si-OH groups of the silica form -Si-O-Si- bonds with the -Si-(O)3- groups of the functionalized triblock molecules of formula (1) of the second hydrogel; and wherein the second hydrogel is in the form of uniformly dispersed particles dispersed within the first hydrogel, the first hydrogel forming a continuous phase; However, the total amount of silica and the functionalized triblock molecule having formula (1) in the second hydrogel is greater than the total amount of silica and the functionalized triblock molecule having formula (1) in the first hydrogel.

[0035] This hydrogel material is two-phase hybrid / composite #1 in Table 1. This hydrogel material therefore has two different hydrogels made up of the same components but in different amounts. The first hydrogel forms the continuous phase of hybrid / composite #1. The second hydrogel first forms a non-flowable hydrogel, and then the physical structure of the second hydrogel is mechanically disrupted to form particles. These particles are then mixed into the continuous phase of the first hydrogel while the first hydrogel is still flowable. Typically, the second hydrogel (i.e., the dispersed phase) contains less aqueous liquid than the first hydrogel.

[0036] According to one embodiment, the total amount of silica and functionalized triblock molecules having formula (1) is at least 20% greater in the second hydrogel than the total amount of silica and functionalized triblock molecules having formula (1) in the first hydrogel.

[0037] When a biologically active agent or analog thereof is encapsulated within this hybrid / composite #1, it is mixed with either the still-flowable second hydrogel, or the still-flowable first hydrogel, or both. If slower delivery is the goal, the drug is mixed with a second hydrogel that is particulate in the finished product. This hydrogel material is therefore particularly suitable when two different release profiles are desired, as it has the potential to deliver drugs at two different rates depending on their location within the components of the hydrogel material.

[0038] According to another embodiment, the hydrogel comprises: a functionalized triblock molecule of formula (1) and a first portion of an aqueous liquid, A particulate form obtained by decomposing a hydrogel of a functionalized triblock molecule of formula (1) and an aqueous liquid into particles, dispersed within a silica sol obtained by mixing a second portion of the aqueous liquid; and - The -Si-OH groups of silica form -Si-O-Si- bonds with the -Si-(O)3- groups of the functionalized triblock molecule of formula (1), thus forming a hydrogel material.

[0039] This hydrogel material is two-phase hybrid / composite #2 in Table 1. The hydrogel material has the same chemical components as the hydrogel materials described above, but has two distinct portions. In this embodiment, the continuous phase is formed by silica and an aqueous liquid, and the dispersed phase is formed by first mixing the functionalized triblock molecule with the aqueous liquid, which allows it to form a non-flowable hydrogel, and the resulting hydrogel is mechanically broken down into particles. These particles are mixed into a continuous phase, resulting in a hydrogel material. When a biologically active agent or similar is added to the hydrogel, it is done by adding the agent while the mixture of functionalized triblock molecules and aqueous liquid is still flowable. Because the silica gel forms the continuous phase, this type of hybrid / complex #2 can be made injectable.

[0040] In this embodiment, the final hydrogel material is formed in the same manner as the first hydrogel, i.e., the chemical reaction results in colloids, the colloids aggregate, and the chemical bonds are of the same type in the first hydrogel and the final hydrogel material.

[0041] In the first and second hydrogels, the components are the same, namely, the functionalized triblock molecule of formula (1), silica, and an aqueous liquid. The amounts given below apply mutatis mutandis to the first and second hydrogels.

[0042] The amount of the functionalized triblock molecule of formula (1) is 0.15 to 21.0 wt % based on the total weight of the hydrogel. Thus, the amount of functionalized triblock molecule of formula (1) can be, for example, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 61.0, 62.0, 63.0, 64.0, 65.0, 66.0, 67.0, 68.0, 69.0, 70. 18.5 or 19.5% by weight, up to 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, 18.0, 19.5 or 21.0% by weight.

[0043] The amount of silica is 0.85 to 4.0 wt % based on the total weight of the hydrogel. Thus, the amount of silica can be, for example, from 0.85, 1.0, 1.2, 1.5, 1.8, 2.0, 2.2, 2.5, 2.7, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, or 3.7 wt. % up to 1.0, 1.5, 1.8, 2.0, 2.2, 2.5, 2.7, 2.9, 3.0, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 wt. % based on the total hydrogel weight.

[0044] The amount of aqueous liquid is 75 to 99% by weight based on the total weight of the hydrogel. Thus, the amount of aqueous liquid may be, for example, 75, 78, 80, 82, 85, 88, 90, 92, or 95% by weight to 80, 82, 85, 88, 90, 92, based on the total weight of the hydrogel. 、9 It can be up to 4, 95, 96, 97, 98 or 99% by weight.

[0045] In this embodiment, one of the hydrogels is in the form of particles, and the particle size of the second hydrogel or the first hydrogel is less than 1 mm. In practice, the most typical particle size is a few tens of micrometers to maintain the fluidity of the material, for example, to pass through a 25-27G needle. Preferably, the particle size is less than 0.3 mm. The particle size may be, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 150, 200, 250, 300, 35 0、5 00, 550, 600, 650, 700, 750, 800 or 850 μm to 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 150, 200, 250, 300, 350 、5 00, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μm.

[0046] In the present hydrogel material, the silica is preferably alkoxysilane-derived silica, more preferably tetraethoxysilane-derived silica.

[0047] The aqueous liquids of the first and second hydrogels are preferably independently selected from water, a mixture of water and ethanol, and a biologically compatible buffer. Examples of such biologically compatible buffers are phosphate buffered saline (PBS), citrate, succinate, acetate, benzoate, and mixtures thereof.

[0048] According to a preferred embodiment, the aqueous liquid is a mixture of water and ethanol, containing 50-95% by weight of water, the remainder being ethanol. Thus, the mixture may contain, for example, 60% to 93% water by weight, the remainder being ethanol. Thus, the amount of water may be from 50, 55, 60, 65, 70, 75, 80 or 85% by weight, up to 60, 65, 70, 75, 80, 85, 90 or 95% by weight of the total weight of the aqueous liquid. The aqueous liquid may be solely water.

[0049] The hydrogel material may further include at least one biologically active agent encapsulated therein. The biologically active agent can be encapsulated in different portions of the hydrogel depending on its structure and the intended use of the hydrogel material. Indeed, the biologically active agent may be encapsulated within the first hydrogel, within at least one of the first and second hydrogels, and / or within the gel of the functionalized triblock molecule of formula (1).

[0050] According to one embodiment, the biologically active agent is selected from the group consisting of an immunomodulatory agent and a therapeutically active agent.

[0051] Encapsulation of biologically active agents can be for a variety of uses, such as delivery (eg, oral or parenteral delivery), administration, desensitization, or protection of the biologically active agent (eg, against heat).

[0052] Thus, the biologically active agent may be, for example, an immunomodulatory agent, such as a vaccine or a hyposensitizer for allergies. Some examples are antigens, viruses, specific antigens, and virus-like particles (VLPs). Some examples of therapeutically active agents, also called drugs, are drug molecules of various sizes, such as peptides, proteins, biological drugs, biopharmaceuticals, biosimilar drugs, biobetter drugs, nucleic acid-based drugs, cells and viral vectors. In vaccines, in vivo studies have shown that when antigens are encapsulated in the hydrogel material, they can be administered without additional adjuvants (as typically used in vaccines).

[0053] The present specification also relates to the use of the hydrogel materials described above for the controlled delivery of biologically active agents. It also relates to the use of such hydrogel materials for the protective encapsulation of biologically active agents. Protective encapsulation can be, for example, to ensure stability of the biologically active agent and / or for protection until controlled release.

[0054] In one particular embodiment, the protective encapsulation is to ensure thermal stability of the biologically active agent. By heat stable, we mean that the encapsulation ensures that the biologically active agent will not be destroyed, denatured, or changed in any way when the product containing the biologically active agent is exposed to temperatures above typical refrigerated storage temperatures (4-8°C). It may also be able to protect biologically active agents during long-term or short-term storage at ambient temperature (20-25°C) or elevated temperatures (>25°C, eg, 35-50°C).

[0055] Additionally, the hydrogel can be used to induce an adjuvant effect in immunizations using biologically active agents, meaning that the hydrogel delivered together with an antigen induces an enhanced immune response compared to administration of the antigen alone.

[0056] The hydrogel material can be made in a variety of ways. One possible method for preparing a functionalized triblock molecule involves the following steps. - reacting L-lactide with polyethylene glycol having a molecular weight of 200 to 30,000 g / mol in the presence of a first catalyst in a molar ratio of polyethylene glycol to L-lactide of 2:1 to 20:1 to obtain a triblock molecule; The resulting triblock molecule is reacted with isocyanatopropyltriethoxysilane in the presence of a second catalyst in a molar ratio of triblock molecule to isocyanatopropyltriethoxysilane of 1:2 to obtain a functionalized triblock molecule.

[0057] The second hydrogel is prepared in the same manner as the first hydrogel described above, except that different amounts are used to obtain a second hydrogel having the same components but different relative amounts. In either case, the goal is to have equal numbers of L-lactide groups at both ends of the polyethylene glycol molecule, so the molar ratio can be any even number within the range above.

[0058] Silica sol can be prepared, for example, by hydrolyzing tetraethyl orthosilicate (TEOS) at pH 2 with stirring. The molar ratio of water to TEOS (R) can be, for example, 98 (R98). After hydrolysis, the silica sol is aged at room temperature for, for example, 115 minutes, and then the pH is adjusted to pH 7.2-7.4 using 0.1 M NaOH.

[0059] TEOS is partially converted to ethanol during the reaction. When the amount of water is in excess compared to TEOS (e.g., R200–R400), the resulting hydrogel material is more fluid and can usually be injected using a fine needle. The obtained silica sol does not contain TEOS.

[0060] The single-phase hydrogel material can be prepared by dissolving the functionalized triblock molecule in an aqueous solution and adding this mixture to a silica sol, after which the mixture is aged to form the hydrogel material.

[0061] Two-phase hybrid / composite hydrogel material #1 (described above in Table 1) can be prepared by dispersing a second hydrogel material into gel particles by introducing shear forces by injecting it through a fine needle.

[0062] To prepare the two-phase hybrid / composite hydrogel material #2, the hydrogel material containing the functionalized triblock molecules was formed into a non-flowing hydrogel, which was then broken down into particles, which were added to the freshly prepared silica sol with mixing.

[0063] Other options for forming the hydrogel into particles include: - Applying pressure to the gel, e.g. by pressing the gel; this results in a paste-like product. - Applying lateral shear forces to the gel; this results in a pasty or liquid product. - Mixing the gel, which is essentially like applying lateral shear forces; using low rotation speeds results in a paste-like product, while using high rotation speeds results in a fluid gel. - Vibration of the gel, such as by vortexing; the original homogeneous gel breaks down into small fragments, which become paste-like. - Uses Turrax mixing for fast disintegration; the gel breaks down into small components, but when the components come into contact with the walls of the receiver, the gel returns to a paste-like state. - Cut the gel with a sharp tool; a paste-like gel will form, the volume will increase, and the material will become electrostatically charged. - Shredding of gel; no paste-like gel is formed, the volume increases and the material becomes electrostatically charged. - Crushing the gel by forcing it through a sieve (the gel needs to be well aged, e.g., for at least 3 days); this results in a porous, fluffy gel, which increases in volume by 4 times and the material becomes electrostatically charged. - Applying mechanical comminution to the gel, for example with a mortar and pestle, or a ball mill or other type of mill.

[0064] [Detailed description of the drawings] Figures 1-5A show schematics of the structures of various intermediate and final hydrogel materials. They should not be construed as exact replicas of scale or structure, as the exact three-dimensional structure at the nanoscale is not precisely known.

[0065] Figure 1 shows a schematic diagram of the structure of a functionalized triblock molecule. In this diagram, LA1 represents lactide and PEG represents polyethylene glycol. In this diagram, R is propyl.

[0066] Figure 2 shows a schematic of silica nanoparticle aggregates in a silica sol. The silica sol contains various structures 1, i.e., aggregates made of silica particles, one of which is shown in enlarged view 2.

[0067] Figure 3 shows a schematic diagram of the structure of a reaction product between silica and a functionalized triblock molecule, according to one embodiment. The silica particles in Figure 2 are shown attached to the ends of the functionalized triblock molecule. The schematic, designated by reference numeral 3, shows the relative sizes of the various portions of the product.

[0068] 4 shows the structure of a hydrogel material according to one embodiment, which is shown to form a network structure.

[0069] FIG. 5A shows the structure of a hydrogel material according to another embodiment, namely, the two-phase hybrid / composite hydrogel #1 described above. Enlarged view 4 shows the substructure of hydrogel #1. FIG. 5B shows the structure of yet another embodiment of a hydrogel material, namely, the two-phase hybrid / composite hydrogel #2 described above, with a close-up view of a portion of hydrogel #2.

[0070] 6-27B show the results of experiments that are discussed in more detail in the Examples section. [Example]

[0071] Materials and Methods Preparation of hydrogel Three different types of hydrogel materials were prepared, as shown in Table 1 above.

[0072] The preparation of the hydrogel material consists of several steps: the first step is the preparation of a linker molecule, followed by the preparation of a primary triblock, and the preparation of a functionalized triblock molecule by functionalizing the triblock silica with isocyanatopropyltriethoxysilane. This functionalized triblock molecule is shown in formula (1) and in FIG.

[0073] On the other hand, silica sol is a preparation of silica sol, as shown in Figure 2 (see Figure 4). Then, a hybrid / composite was prepared from the silica sol and the functionalized triblock as shown in Figure 3, and the hybrid / composite hydrogel was formed (shown in Figure 4).

[0074] Two-phase hybrid / composite hydrogels #1 and #2 were prepared by dispersing other hydrogel particles and then mixing the dispersed hydrogel particles into another hydrogel that served as the continuous phase, as shown in Figures 5A and 5B.

[0075] (Preparation of sequential hybrid / composite hydrogels and embedding of norovirus P-particles / proteins) The formation of the triblock was initiated by carrying out a ring-opening polymerization (ROP)-like reaction of L-lactide (Sigma Aldrich) to polyethylene glycol (PEG, Sigma Aldrich), catalyzed by tin(II) 2-ethylhexanoate (also serving as the initiator), to prepare a linker molecule (PEG end-capped at both ends with L-lactide). The operation was carried out as a solvent-free bulk polymerization in a Schlenk line system under an inert atmosphere of N2. The required amount of midblock (polyethylene glycol, PEG, 200 g / mol) was calculated according to a molar ratio of 2:1 (PLA:PEG). The PEG was added, after which the temperature was increased to 160°C with gentle stirring. The mixture was heated until the contents were melted. Tin(II) 2-ethylhexanoate (Sigma Aldrich) at a molar ratio of 0.05 (catalyst:PEG) was pipetted into the melt and the vessel was sealed. The reaction was allowed to proceed for 90 minutes at 160° C. After the reaction, the product was cooled to ambient temperature. The product was purified in a binary solvent system of chloroform and hexane (Sigma Aldrich). The hexane supernatant was decanted and the chloroform was evaporated in two steps. First, chloroform was evaporated in a rotary evaporator (IKA RV10) at a vacuum of 450 mbar for 15 min at 40 °C. In a second step, the last traces of solvent were evaporated with a vacuum pump. The resulting triblock was warmed to +60° C. in a water bath and the pressure was reduced to p<0.1 mbar with a diffusion pump (Vacuubrand RZ 2.5). The evaporating material was captured in a solvent trap immersed in liquid nitrogen (N2). This procedure was continued until no more bubbles formed above the melt.

[0076] The prepared triblock was then reacted with isocyanatopropyltriethoxysilane (IPTS, Sigma Aldrich) to produce a functionalized triblock molecule by dissolving the triblock, IPTS, and dibutyltin dilaurate (catalyst, Alfa Aesar) in tetrahydrofuran (THF, Sigma Aldrich) in a molar ratio of 1:2:0.05. The mixture was heated to 60°C under an inert atmosphere using a Schlenk line system as described in the triblock preparation step above. The reaction was allowed to proceed for 1 hour. The THF was evaporated using a rotary evaporator (IKA RV10) at 357 mbar vacuum for 10 min at 40 °C. The resulting functionalized triblock molecule was then dissolved in a binary solvent system of hexane and chloroform. Further purification steps of the functionalized triblock molecule were carried out exactly as described above in the triblock preparation section.

[0077] Next, typical examples of other steps in the preparation process of hybrid / composite hydrogels are described. Several different formulations were prepared by varying the molecular weight of the polymer (i.e., the molecular weight of the polyethylene glycol) and the amounts of P-particles, functionalized triblock molecules, water, and silica, while the preparation process remained the same except for slight variations in the mixing, aging, and gelation times.

[0078] The next step after the synthesis of the functionalized triblock molecule was the preparation of a silica sol. Tetraethyl orthosilicate (TEOS, Sigma-Aldrich) was used as the silica precursor. TEOS was hydrolyzed with stirring at pH 2 (adjusted using 0.1 M HCl, Merck Titripur). The molar ratio of water to TEOS (R) was 98 (R98). After hydrolysis, the silica sol was aged at room temperature for 115 min, after which the pH was adjusted to pH 7.2–7.4 using 0.1 M NaOH (Merck Titripur). The resulting silica sol was filtered through a sterile 0.45 μm PES membrane syringe filter.

[0079] 200 mg of functionalized triblock molecules (containing 200 g / mol of PEG) were dissolved in 50 ml of phosphate-buffered saline (PBS, Sigma Aldrich) containing norovirus P-particles at a concentration of 200 micrograms / ml (200 ppm). This mixture was then added to 50 ml of R98 silica sol (aged at pH 7.2-7.4 for approximately 3 minutes) to give a P-particle concentration of 100 ppm in the resulting sol. The sol (1 ml) was transferred to a syringe within 2-5 minutes before the sol (total volume approximately 100 ml) turned into a non-flowing gel (gelling time varies depending on the formulation details). Evaporation of the aqueous solution (water and ethanol) was minimized during the preparation and pH adjustment steps by covering the container with plastic film. The resulting water-to-TEOS ratio (R) was 217 (R217), corresponding to 1.5 wt% silica in the final hydrogel material. The final concentration of functionalized triblock molecules was 0.2 wt%, and the P-particle concentration was 100 ppm, i.e., 0.1 mg per ml of final hydrogel material. In the above example, the final code for the hydrogel material is R217-0.2, which indicates the content of both silica and functionalized triblock molecules in the final material. The composition of the R217-0.2 hydrogel is detailed in Table 2.

[0080] [Table 2]

[0081] The actual hydrogels consisted of functionalized triblock molecules, silica, water, and ethanol, and contained encapsulated antigens. In addition, the hydrogels may contain residual substances, namely NaCl, encapsulated within their structure. Different hydrogel materials corresponding to different component concentrations were prepared (R217-0.15, R328-0.2, R328-0.4, R328-0.8, R40-0.2). Higher molar mass PEGs (2000 g / mol and 6000 g / mol) were also tested for sequential hybrids / conjugates of R217, but their injection properties were not optimal, so more extensive studies on hybrids / conjugates were carried out only with 200 g / mol PEG in the triblock. However, such materials (i.e., high molar mass PEG) are useful for non-injectable delivery purposes, as their properties are otherwise similar to those prepared and tested below.

[0082] (Preparation of two-phase hybrid / composite hydrogels and embedding of norovirus P-particles / proteins) The above functionalized triblock molecules are part of two-phase hybrid / composite hydrogels #1 and #2 (listed in Table 1 above). In two-phase hybrid / composite hydrogel #1, the dispersed hydrogel fragments consist of the same solid phase as the continuous phase, but with a different amount of water. In two-phase hybrid / composite hydrogel #2, the continuous hydrogel is inorganic silica gel and the dispersed component is organic hydrogel. The general structure of the two-phase hydrogel is shown in Figures 5A and 5B. The actual hydrogel is composed of functionalized triblock molecules, silica, water, and ethanol, and the antigen is encapsulated. In addition, the hydrogel may contain residual substances, namely NaCl, encapsulated within its structure.

[0083] In two-phase hybrid / composite hydrogel #1 (Table 3), both components are of the same type, containing alkoxysilane-functionalized triblock molecule-silica hybrid / composite (organic-inorganic hybrid / composite), and the only difference is the amount of water used in the hydrogel. Different two-phase hydrogels with different water-to-TEOS molar ratios (R40, R217, and R328) and functionalized triblock molecule contents (0.15 and 0.2 wt % for R217, 0.2, 0.4, and 0.8 wt % for R328, and 0.2 wt % for R40) were prepared, but R217-0.2 / R40-0.2 was selected for more rigorous characterization due to its superior rheological properties and injectability. The former component (here, R217-0.2) represents a continuous hydrogel phase, and the latter component (here, R40-0.2) represents a hydrogel dispersed in the continuous hydrogel phase. Norovirus P-particles were embedded in the dispersed hydrogel only or in both dispersed and continuous hydrogels in the corresponding manner described above. In addition, monomeric green fluorescent protein (eGFP) was used as an alternative encapsulating agent for R217-0.2. Dispersion hydrogel R40-0.2 was prepared by first allowing this system to form a non-flowable gel (ensuring effective embedding of P-particles). Immediately after hydrogel formation, the non-flowable R40-02 was dispersed into gel particles by introducing shear force through injection with a fine needle (18G). This step was carried out rapidly (within 1–2 min) to avoid evaporation of the aqueous liquids (water and ethanol) from the system. The main function of continuous hydrogels is to act as an injection matrix, i.e., to provide two-phase systems with rheological properties suitable for injection through thin needles, but they can also be used as part of a controlled release system to achieve different release rates, e.g., biphasic release.

[0084] Table 3 shows the composition of two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) containing encapsulated antigen and residue (A: antigen in both R217 and R40 at a mass ratio of 25:75, and B: antigen in R40 only).

[0085] [Table 3]

[0086] In two-phase hybrid / composite hydrogel #2, the continuous component was a silica hydrogel, and the dispersed component was a hydrogel composed of alkoxysilane-functionalized triblock molecules and water. Four different hydrogel formulations were prepared with different weight ratios of silica, siloxane-functionalized triblock molecules, and water (Table 4). Tetraethyl orthosilicate (TEOS, Sigma Aldrich) was used as the silica precursor. TEOS was hydrolyzed at pH 2 (adjusted using 0.1 M HCl) under stirring. The molar ratio of water to TEOS (R) was 220 (R220) for each formulation. Additional ethanol was used in the aqueous solution to establish a homogeneous system with silica, functionalized triblock molecules, and water (the formation of silica sol from TEOS results in the formation of the by-product ethanol). The pH of the resulting silica was adjusted to pH 5-6 (using 0.1 M NaOH) before adding the organic hydrogel moiety containing siloxane-functionalized triblock molecules and water. The preparation of the dispersed organic hydrogel began by first forming a non-fluid hydrogel in this system (ensuring effective embedding of the P-particles). After hydrogel formation, the structure was dispersed into the particles by grinding the hydrogel into hydrogel particles. To avoid water separation, the addition of the dispersed organic hydrogel particles to the freshly prepared silica sol was carried out quickly within 1–2 min, and the whole system was homogenized using an Ultra-Turrax T25 (with stator S25N-18G). The method of dispersing the organic hydrogel is different from that of two-phase hybrid / composite hydrogel #1 because of the different amount of solids in two-phase hybrid / composite hydrogel #2, which is higher. The mixture of silica gel and organic hydrogel particles turns into a non-flowing hydrogel in about 30 minutes, and mixing is performed prior to hydrogel formation to avoid settling of the organic hydrogel portion.

[0087] Table 4 shows the various compositions of two-phase hybrid / composite hydrogel #2 containing encapsulated antigen and residue.

[0088] [Table 4]

[0089] Norovirus P-particles were embedded in two-phase hybrid / composite hydrogel #2 as described above (i.e., the P-particle solution was added to the system while it still had a flowing, liquid-like structure prior to hydrogel formation), but in this case, they were dispersed only within the organic hydrogel. If desired, P-particles or other biologically active agents can again be embedded within both the dispersed and continuous hydrogels.

[0090] (Production and characteristics of eGFP) Monomeric green fluorescent protein (eGFP) was produced using recombinant Escherichia coli (the gene encoding monomeric green fluorescent protein was transformed into E. coli) as the production organism. The protein was purified using standard molecular biology methods.

[0091] The following procedure was used to embed eGFP into the hydrogel. Protein stocks manufactured in 2017 and stored at -20°C were used in the experiments. Prior to the above experiments, the protein content was measured using μBCA (microBCA Protein Assay Kit, Microplate Procedure, Thermo Scientific™). The concentration was 2.2 mg / ml. The molar mass of eGFP was determined to be approximately 40 kDa by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

[0092] Dissolution experiment (Release of P particles and eGFP and biodegradation of hybrid / composite hydrogels) Dissolution experiments, i.e., release of P-particles and dissolution of hybrid / composite hydrogels (R217-0.2 and R217-0.2-R40-0.2), were carried out in a shaking bath at 37°C in either PBS or tris(hydroxymethyl)aminomethane (TRIS) buffered to approximately pH 7.4. PBS was used for the release of P-particles, and TRIS was used for the dissolution of inorganic-organic hybrids / complexes. Dissolution experiments were carried out under sink conditions (to ensure free dissolution without hindering the dissolution rate by dissolution products), which actually means sink conditions for the inorganic part (amorphous silica) of the hybrid / composite hydrogel. The dissolution medium was periodically refreshed at all sampling time points to maintain in-sink conditions (concentration of dissolved amorphous silica below 30 ppm). Three replicates were collected at each time point.

[0093] P-particles and eGFP release were analyzed by total protein assay with a microBCA protein assay kit (Microplate Procedure, Thermo Scientific™) for colorimetric detection at 562 nm with a spectrophotometer (Hidex Sense Microplate Reader). The dissolution rates of the hybrid / composite hydrogels (continuous hybrid / composite and two-phase hybrid / composite #1) were estimated by measuring the dissolution of silica as a function of time using a GF-AAS (Shimadzu 6650F, GFA-EX7), which corresponded to total solid contents of the hybrid / composite hydrogels of 3.5% (for two-phase hybrid / composite hydrogel #2-D)–82.5% (for two-phase hybrid / composite hydrogel #1-B). Prior to the analysis, the liquid samples containing the P-particles were concentrated. Concentration was performed because dissolution measurements were performed under sink conditions (for silica), resulting in very diluted samples (and sink conditions were used to find the actual mechanism of release, taking into account both biodegradation and diffusion-related components in the release, and to observe actual differences between the different material versions). The settling limit for silica was approximately 30 ppm, so the maximum concentration of encapsulated material correlates to the same limit depending on the loading rate. At a loading of 1% of the total silica mass, the maximum concentration of encapsulated material in the dissolved sample is only 0.3 ppm. Samples were concentrated by either centrifugal membrane filtration (Eppendorf Centrifuge 5810 R, 8000-10000g, 15-30 minutes, using Amicon® Ultra-15 centrifugal filters Ultracel®-3K) or freeze-drying (Christ Epsilon 1-6D, loading and maximum freezing temperature at -35°C, followed by a gradual increase in temperature to -15°C for main drying, and final drying at -10°C for 23 minutes and 20°C for 20 minutes), after which the freeze-dried P-particles were redispersed in a smaller volume of liquid.

[0094] Dissolution of P-particles from hydrogel R217 for particle composition and integrity studies To examine whether the released antigen remained intact after being kept at different temperatures, hydrogels containing 0.5 mg / ml P-particles were dissolved in a 10 kDa cutoff dialysis cassette (Slide-A-Lyzer, ThermoFisher). First, the hydrogel was removed from the syringe, weighed, and transferred to a dialysis cassette, which was dialyzed in 0.8 L of PBS changed five times over a period of 1 week at room temperature. Next, after verifying that the cassette was free of gel residue, the solution from the cassette was transferred to microcentrifuge tubes, aliquoted, and stored at −20°C for analysis.

[0095] (Release mechanism experiment) The release mechanism of P-particles was investigated in a shaking bath at 37 °C in silica-saturated PBS or tris(hydroxymethyl)aminomethane (TRIS) buffered to approximately pH 7.4. Silica-saturated media was prepared by dissolving amorphous sol-gel-derived silica flakes in the media until a silica saturation level (approximately 130 ppm) was reached. When dissolution of the bulk of the hybrid / composite hydrogel (silica) was prevented, P-particles were released primarily by diffusion alone. The diffusion results were then compared with release experiments carried out under sink conditions, where both matrix biodegradation and diffusion could occur simultaneously. The release mechanism of sequential hybrids / complexes containing R217-based encapsulated P-particles and eGFP was investigated.

[0096] Rheological measurements and injection experiments Rheological measurements were performed using a modular rheometer (Anton Paar MCR 104). Samples were injected from a 1 ml syringe (Becton Dickinson) through a 25 G needle (Becton Dickinson Microlance) onto a 20 mm plate-plate geometry. Amplitude sweep measurements were performed at a constant 6.28 rad / s angular frequency with strain values ​​ranging from 0.01 to 10%. Frequency sweeps were performed at frequencies from 0.01 to 100 rad / s at the constant strain defined by the amplitude sweep measurements.

[0097] The injection performance of the prepared hybrid / composite hydrogel materials was investigated. Tests were conducted on the continuous hybrid / composite and two-phase hybrid / composite #1 hydrogels. The material was injected with normal force from a 1 ml syringe (Becton Dickinson) through a 25 G needle (Becton Dickinson Microlance). A line of hydrogel was injected into a plastic tray and the results were visually inspected. Smoothness of injection, signs of phase separation, apparent homogeneity, and compatibility with the lysis buffer were recorded and evaluated.

[0098] 2.4 Production and purification of norovirus P-particles His-tagged norovirus P-particles were produced in E. coli BL21 star cells as described by Koho et al. (Journal of Virological Methods 179 (2012) 1-7). Pelleted bacterial cells were lysed in lysis buffer (50 mM NaHPO, 600 mM NaCl, 10 mM imidazole, pH 8.0) using an EmulsiFlex®-C3 homogenizer (Avestin Inc.). The cell lysate was clarified at 10,000 × g for 30 minutes at 4 °C. The clarified lysate was used for affinity purification using nickel-charged Sepharose (Merck, HisTrap FF Crude). The purified P-particles were dialyzed against PBS and then sterile-filtered for analysis and further use. The concentration of P-particles was measured by BCA assay (Pierce).

[0099] Dynamic light scattering analysis Dynamic light scattering (DLS) analysis of P-particles was performed using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Worcestershire, UK). Hydrodynamic diameters were determined using three 10 × 10-second data sets in PBS at 25°C. Samples were also subjected to stepwise heating as described by Koho et al. (Antiviral Research 104 (2014) 93-101). Starting at 25°C, each sample was heated in 5°C increments and allowed to equilibrate at each temperature for 5 minutes before analysis. Samples were heated to a final temperature of 90°C and then cooled to 25°C. To investigate the integrity and composition of P-particles dissolved in PBS from the continuous hybrid / composite hydrogel R217-0.2, they were analyzed using DLS at 25°C.

[0100] Endotoxin measurement To confirm that endotoxin had been removed from the purified antigen, endotoxin levels in the P-particles were measured using the ToxinSensor™ Gel Clot Endotoxin Assay Kit (GenScript) according to the manufacturer's instructions.

[0101] Production and purification of norovirus VLPs Norovirus (NoV) GII capsid virus-like particles (VLPs) from GII.4 (1999, acc. no. AF080551), GII.4 New Orleans (NO; 2010, acc. no. GU445325), GII.4 Sydney (Syd; 2012, acc. no. AFV08795.1), GII.12 (1998, acc. no. AJ277618), and GII.17 (2015, acc. no. BAR42289) were obtained as previously described elsewhere (Huhti et al. Arch Virol 2010 155; Blazevic et al. Vaccine 2011 29; Malm et al. Clinical and vaccine immunology 2015 164). It was produced in Sf9 insect cells by the Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA) and purified by sucrose gradient ultracentrifugation, as described in detail in 22 ). These VLPs were used as antigens in in vitro immunogenicity assays.

[0102] Animal immunization Pathogen-free female 6-week-old BALB / c OlaHsd mice (Envigo, Horst, The Netherlands) were randomly divided into eight groups (Gr I-VIII, 3 or 5 mice / experimental group) and allowed to acclimate under specific control conditions for 1 week before the start of the experiment. Animals were immunized with NoV P-particles diluted in sterile PBS (Lonza, Verviers, Belgium) at two 10 μg doses or one 20 μg dose, or formulated sequential hybrid / complex R217-0.2 or two-phase hybrid / complex #1R217-0.2 / R40-0.2 (A and B, Table 3 ) hydrogels. Test articles were administered by subcutaneous (sc) injection (100 μl volume) into the right flank on study weeks 0 and 3. Table 5 shows the vaccine formulations, injection doses, and immunization methods used. Control groups received only P-particles formulated with R217-0.2 or R217-0.2 / R40-0.2 hydrogels, or Al(OH)3 (Alhydrogel; InvivoGen, San Diego, CA). Immunizations were performed under general anesthesia with inhalation of isoflurane (Attane vet, Vet Medic Animal Health Oy).

[0103] To examine the kinetics of serum antibody responses, blood samples were collected by tail bleed at study week 0 (pre-bleed, non-immune serum) and study week 3. Whole blood and feces were collected at the time of sacrifice (5 or 6 weeks) and processed according to published procedures ("Norovirus VLPs and rotavirus VP6 protein as a combined vaccine for childhood gastroenteritis" Blazevic et al, Vaccine Oct 19;29(45):8126-33.; "A comparison of immunogenicity of norovirus GII-4 virus-like particles and P-particles", Tamminen et al., Immunology 2012 Jan;135(1):89-99). Experimental procedures were carried out in accordance with the rules and guidelines of the Finnish National Experimental Board (permit number ESAVI / 10800 / 04.10.07 / 2016). Every effort was made to minimize animal suffering. Animal welfare was monitored throughout the study.

[0104] Table 5 shows the antigen composition, injection amount and immunization method.

[0105] [Table 5]

[0106] antigen-specific antibody reaction Antibody responses against P-particles were determined by measuring IgG and IgG subtype levels in serum samples from individual mice by ELISA. The ELISA procedure used was similar to that previously published by our laboratory (Blazevic et al. Vaccine 2011 29; Tamminen et al. Immunology. 2012 135(1):89-99), and therefore only a brief summary is provided below. Half-area polystyrene plates (Corning Inc., Corning, NY) were coated with 50 ng of NoV P-particles per well. Antigen-specific antibodies in sera diluted 1:200 or in serial two-fold dilutions were detected with HRP-conjugated anti-mouse IgG (Sigma-Aldrich), IgG1 (Invitrogen), or IgG2a (Invitrogen) in combination with SIGMA FAST OPD substrate (Sigma-Aldrich). The endpoint titer was defined as the reciprocal of the highest sample dilution with an OD490 above the cutoff value (>0.1 OD490 units).

[0107] Antibody Avidity The avidity of NoV GII.4-specific IgG antibodies was assessed in 1:200 diluted serum samples according to the ELISA method described above in 1.5 (Tamminen et al. Immunology. 2012 135(1):89-99), but with an additional urea treatment to remove low-avidity antibodies. Plates were coated with 50 ng of NoV GII.4 VLPs. Results were expressed as an avidity index: (OD490 with urea / OD490 without urea) × 100%.

[0108] Cross-reactive antibodies Cross-reactive NoV-specific IgG antibodies were detected by ELISA as described above, except that plates were coated with 50 ng of heterologous NoV VLPs per well, including GII.4 NO, GII.4 Sydney, GII.12, and GII.17 VLPs. A serum dilution of 1:200 was used in the assay.

[0109] Blocking antibodies The ability of the induced antibodies to prevent the binding of NoV VLPs to the HBGA receptor was assessed in a blocking assay using PGM type III (Sigma Chemicals) as the HBGA source (Lindesmith et al. J Virol 2012; 86:873-83) according to previously published procedures (Malm et al. Clin Exp Immunol 2017;189(3):331-41). Briefly, a mixture of preincubated GII.4 VLPs and serially diluted serum was added to a PGM-coated microwell plate. Bound VLPs were detected with a combination of human NoV GII.4 antiserum and HRP-conjugated anti-human IgG according to the receptor binding assay described above in 1.2. The results were expressed as a blocking index: 100% - [(OD490 sample / OD490 maximum binding) × 100%].

[0110] Mucosal IgG response For detection of mucosal IgG antibodies, serially diluted 10% fecal suspensions were tested by the ELISA method described in 1.5 above. Plates were coated with 50 ng of NoV GII.4 VLPs.

[0111] Results and Discussion In vitro lysis and antigen release The in vitro dissolution results of antigen (P-particle) release rate and silica dissolution rate of different hybrid / composite hydrogels are shown in Figures 6 to 8 . Figure 6A shows the antigen (P-particle) release rates for the continuous hybrid / composite R217-0.2 and two variations of the two-phase hybrid / composite hydrogel #1: R217-0.2 / R40-0.2A (antigen (AG) present in both the R217-0.2 and R40-0.2 hydrogels) and R217-0.2 / R40-0.2B (antigen (AG) present only in the R40-0.2 hydrogel) after storage in syringes placed in aluminum foil bags at room temperature (25 °C) for 4 and 330 days. Figure 6B shows the cumulative release of silica under the same conditions.

[0112] FIG. 7 shows the cumulative release of antigen (P-particles) and silica dissolution for two-phase hybrid / composite hydrogel #2C. The weight ratio of the released amounts of silica and P-particles at different time points is shown on the second Y-axis on the right.

[0113] Figure 8 shows the cumulative release of antigen (P-particles) and dissolution of silica from two-phase hybrid / composite hydrogel #2D. The weight ratio of the released amount of silica to that of P-particles at different time points is shown on the secondary Y-axis on the right.

[0114] The release results for R217-0.2 containing encapsulated eGFP show that silica dissolution and eGFP release occur simultaneously, although there are some differences in the amount of encapsulated eGFP. With 20 μg of eGFP in 100 μl of R217-0.2, both the eGFP release rate and the silica dissolution rate are slightly slower at first, but reach approximately 100% at almost the same time.

[0115] Figure 9 shows the cumulative release of eGFP and silica dissolution in 100 μl of R217-0.2 hydrogels containing 10 μg of eGFP. The weight ratio of the released silica to the released eGFP at different time points is shown on the secondary Y-axis on the right.

[0116] Figure 10 shows the cumulative release of eGFP and silica dissolution in R217-0.2 hydrogels containing 20 μg of eGFP per 100 μl of hydrogel. The weight ratio of silica to eGFP release at different time points is shown on the secondary Y-axis on the right.

[0117] To investigate the encapsulation efficiency of the materials, the cumulative release of eGFP from silica-saturated dissolution media from R217-0.2 containing 10 μg and 20 μg of eGFP per 100 μl of hydrogel was measured. As shown in Figures 9 and 10 , the rate at which eGFP diffused from the hydrogel during the first 5 h was approximately 50% slower than under sink conditions. The release rate slowed slightly after 24 hours, leveling off at 25% for the 10 μg / 100 μl material and 35% for the 20 μg / 100 μl version.

[0118] FIG. 11 shows the cumulative diffusion of eGFP from R217-0.2 at an eGFP concentration of 10 μg / 100 μl of hydrogel.

[0119] FIG. 12 shows the cumulative diffusion of eGFP from R217-0.2 at an eGFP concentration of 20 μg / 100 μl of hydrogel.

[0120] As the concentration of eGFP at 24 or 25 hours is less than 0.5% of the stock concentration of the protein, it is unlikely that eGFP interferes with its own release in terms of saturation. Furthermore, during diffusion measurements, when the sample volume was 10 μg / 100 μl, the release rate of eGFP reached a plateau after 24 h and remained somewhat stable, while the same was observed for the 20 μg / 100 μl version, which still appeared to be stable. Comparison with dissolution in the sink suggests that while some diffusion occurs, the entire eGFP payload is not effectively released unless degradation of the material occurs.

[0121] Rheology Figure 13 shows the damping coefficient (G'' / G') for the continuous hybrid / composite hydrogel R217-0.2 and for the two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) at 4 and 28 days after storage in a syringe enclosed in an aluminum foil bag at room temperature (25 °C).

[0122] Figure 14 shows the attenuation coefficient (G" / G') for the continuous hybrid / composite hydrogel R217-0.2 and the two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) after 360 days of storage at room temperature (25 °C). The syringes were isolated in aluminum foil bags. The samples were subjected to a three-part measurement in which the first step was a frequency sweep, followed by a rotational measurement (shear rate ramp 1-100 1 / s), and then a frequency sweep measurement again.

[0123] FIG. 15 shows the dynamic viscosity of the continuous hybrid / composite hydrogel R217-0.2 and the two-phase hybrid / composite hydrogel #1 (R217-0.2 / R40-0.2) after 4 days of storage at room temperature (25° C.).

[0124] FIG. 16 shows the dynamic viscosity of continuous hybrid / composite hydrogel R217-0.2 and two-phase hybrid / composite #1 (R217-0.2 / R40-0.2) after 360 days of storage in sealed aluminum foil bags at room temperature.

[0125] Rheological measurements show that the investigated hybrid / composite hydrogels retain their rheological properties very well even after 28 days of storage in syringes placed in aluminum foil bags at 25 °C. The damping coefficient (also called loss tangent or loss factor) is the ratio of the loss modulus to the storage modulus (G'' / G') and describes the viscoelastic properties of a hybrid / composite hydrogel at rest (e.g., in a syringe). The damping coefficient results show that the hydrogels retained their structure during storage and there was no significant change in properties after 28 days of storage at 25 °C (Figure 13). Dynamic viscosity (Figure 16), which simulates injection from a syringe, showed clear shear thinning for both hydrogels investigated after 4 days of storage at 25°C, with properties remaining relatively unchanged over a 1-year period (Figures 14 and 17). The differences occur primarily in the dynamic viscosity. Overall, the minimum viscosity at low shear rates is significantly reduced, and the material shears more slowly compared to the D4 and D28 cases. The overall behavior remains unchanged, and the material was able to retain its rheological properties during storage.

[0126] Injectability In addition to rheological measurements, the injectability and stability of the hybrid / composite hydrogels were evaluated by injecting the material from a 1 ml syringe through a 25 G needle. Injectability or the possibility of phase separation was evaluated for the continuous hybrid / composite hydrogel embedded with P-particles R217-0.2(AG) and two variations of the two-phase hybrid / composite hydrogel #1 embedded with P-particles R217-0.2(AG) and P-particles (R217-0.2-R40-0.2(AG) and R217-0.2(AG)-R40-0.2(AG)) after storage in a syringe placed in an aluminum foil bag at room temperature (25°C) for 4, 28, 180, and 330 days, according to a scale of 0 to 5 (Table 6). The criteria for injection smoothness are defined as follows: 5 = very smooth, 4 = mostly smooth, 3 = fairly smooth, 2 = partly smooth, 1 = uneven, not smooth at all, 0 = not pourable. The criteria for phase separation are defined as follows: 5 = no phase separation, 4 = minimal phase separation, 3 = phase separation but reversible after injection, 2 = phase separation, irreversible after injection, 1 = obvious phase separation, 0 = no gel at all.

[0127] Table 6 shows the pouring experiments for pour smoothness and phase separation at various time points after storage at 25° C. Scores are the average of three replicate pours.

[0128] [Table 6]

[0129] The injection experiments indicate that the smoothness observed on day 4 did not change during storage, and the small changes observed between the continuous hybrid / composite hydrogel and the two-phase hybrid / composite hydrogel #1 originate from differences in structure, i.e., the individual gel particles present in both versions of the two-phase hybrid / composite hydrogel #1. Although they are all injectable, the results suggest that R217-0.2(AG)-R40-0.2(AG), which contains encapsulated P-particles in both the continuous and dispersed phases, is somewhat heterogeneous in nature but still injectable. The amount of encapsulated antigen was so small that it is unlikely that it would have such an effect, but rather a minor difference in the preparation process (addition of P-particles in R217-0.2), which highlights the need for strict control of the preparation process. The minimal or even phase separation observed is related to the initial appearance of a small amount of water at the start of injection, or the observation of a hydrogel mass associated with injection, which however reconsolidates to form a consolidated hydrogel mass after injection.

[0130] Aggregate formation of P-particles in high-temperature solutions As previously shown (Journal of Virological Methods 179 (2012) 1-7), norovirus P-particles have a diameter of approximately 15-17 nm in solution when analyzed by DLS (not shown). To investigate the thermal stability of P-particles, we first determined the temperature at which they aggregate in solution. This analysis revealed that rapid temperature-induced aggregation occurs above 50 °C, and all P-particles form aggregates ranging from 300 to 830 nm at 55 °C (Figure 17). Based on these results, long-term storage and dissolution experiments were performed using the following temperatures: RT, 37°C, and 50°C.

[0131] Figure 17 shows the thermal stability of P-particles measured by DLS. In Figure 17A, the thermal stability of P-particles at different temperatures is 15 17A and 17B show the difference in the volume distribution of P-particles measured at 50° C. and 55° C. Both Figures 17A and 17B show the average of three individual measurements.

[0132] Particle integrity after dissolution The integrity of the particles dissolved from the hydrogels was examined by DLS. During the 28-week follow-up, virtually all particles released from the hydrogels kept at room temperature remained at the expected size, while particles released from the hydrogels kept at 37°C remained within the expected radius range of 15–17 nm by 19 weeks (Figure 18). Control samples in solution stored at RT remained intact, while those stored at 37°C began to break down after 6 weeks and were completely broken down within 16 weeks. Furthermore, both the hydrogel and solution samples kept at 50°C broke down within 4 weeks or 2 weeks, respectively.

[0133] Figure 18 shows the dynamics of P-particles released from the continuous hybrid / composite hydrogel R217-0.2 (Figure 18A) or from control particles kept in solution at the indicated temperature as determined by DLS (Figure 18B). 15 The total volume of the aggregate is shown in nm.

[0134] TEM images Transmission electron microscopy images show that the hydrogel protects the P-particles longer at 37° C. than control particles kept in solution at the same temperature.

[0135] TEM shows that the control P-particles have a typical shape and the expected size of about 17–20 nm ( Figure 19 , top panel). P-particles remain stable in solution at RT for 20 weeks (Figure 19, bottom left panel), but appear to disappear after 12 weeks in solution at 37°C (Figure 19, center panel). P-particles dissolved from the hydrogel after storage at RT and 37°C appear intact after 19 weeks (Figure 19, bottom center panel).

[0136] Endotoxin levels Endotoxin levels were determined to confirm endotoxin removal from the antigen: the antigen was free of bacterial endotoxins (<0.012 EU / 10 μg protein), and no residual impurities were detected in the purified P-particles.

[0137] Development of serum IgG antibodies To examine the potential of this hydrogel to function as an adjuvant / delivery system, mice were immunized with two 10 μg doses of P-particles alone or embedded in sequential hybrid / composite R217-0.2 hydrogels, or with one 20 μg dose of P-particles alone or embedded in two-phase hybrid / composite #1 R217-0.2 / R40-0.2 hydrogels. For comparison, one of the experimental groups received P-particles formulated with Al(OH)3. Figure 20 shows the development of serum IgG antibodies to P-particles at study weeks 0, 3, and 5 or 6. Results showed that immunization with a single dose of P-particles only elicited a very strong immune response when the antigen was co-administered with R217-0.2 hydrogel or R217-0.2 / R40-0.2A hydrogel (25 wt-% antigen in R217-0.2). This indicates that the increased antibody response 3 weeks after single immunization was due to the R217-0.2 component, not the R40-0.2 component. A second administration of P-particles containing R217-0.2 hydrogel significantly boosted the already induced response.

[0138] Figure 20 shows the kinetics of serum IgG antibodies in mice after immunization with two 10 μg doses of P-particles (pp) alone or formulated with R217-0.2 hydrogel (Figure 20A), or one 20 μg dose of P-particles alone or formulated with R217-0.2 / R40-0.2 hydrogel (Figure 20B). Control mice received P-particles containing Al(OH)3 or antigen-free hydrogel. Group mean OD values ​​for tail blood samples and terminal serum at the indicated study weeks are shown, with immunization points indicated by arrows.

[0139] Furthermore, the geometric mean titers were significantly higher (reciprocal titers >4.2 log) in the groups of mice administered P-particles containing R217-0.2 or R217-0.2 / R40-0.2A hydrogel (25% by weight of antigen in R217-0.2). 10 ), co-administration resulted in levels 18-fold or 10-fold higher than those observed with P-particles alone (FIG. 21). This indicates that R217-0.2 increases the magnitude of the antigen-specific IgG response. However, the observed effect of R217-0.2 on P-particles is similar to that of Al(OH)3 hydrogel (Figures 21 and 22). Negative control mice had no response to P-particles.

[0140] Figures 21A and 21B show serum IgG endpoint titrations in mice immunized with two 10 μg doses of P-particles (pp) alone or formulated with R217-0.2 hydrogel (Figure 21A), or one 20 μg dose of P-particles alone or formulated with R217-0.2 / R40-0.2 hydrogel (Figure 21B). Control mice received P-particles containing Al(OH) or antigen-free hydrogel. Shown are the mean ODs for the groups.

[0141] Serum antigen-specific IgG1 and IgG2a subtypes Analysis of antigen-specific IgG subtypes, IgG1 (characteristic of a Th2 response) and IgG2a (characteristic of a Th1 response), showed that only P-particles formulated with the sequential hybrid / composite hydrogel R217-0.2 or Al(OH)3 induced a mixed immune response, i.e., Th2- and Th1-type (Figures 22 and 23). Although each P-particle formulation induced a Th2 immune response, coadministration of P-particles with R217-0.2, or two-phase hybrid / composite #1 R217-0.2 / R40-0.2A hydrogel (25% by weight of antigen in R217-0.2), or Al(OH)3 produced higher levels of IgG1 compared with administration of P particles alone (Figure 22). Rather, only P-particles co-administered with R217-0.2 hydrogel or Al(OH) 3 induced a moderate Th1 immune response (FIG. 23). These results suggest that R217-0.2 improves the quality of the immune response and preferably functions as a Th2 adjuvant similar to Al(OH)3.

[0142] Figure 22 shows the endpoint titration of serum IgG1 in mice after immunization with two 10 μg doses of P-particles (pp) alone or formulated with R217-0.2 hydrogel (Figure 22A), or one 20 μg dose of P-particles alone or formulated with R217-0.2 / R40-0.2 hydrogel (Figure 22B). Control mice received P-particles containing Al(OH)3 or hydrogel without antigen. Shown are the mean ODs of the groups.

[0143] Figure 23 shows the endpoint titration of serum IgG2a in mice after immunization with two 10 μg doses of P-particles (pp) alone or formulated with R217-0.2 hydrogel (Figure 23A), or one 20 μg dose of P-particles alone or formulated with R217-0.2 / R40-0.2 hydrogel (Figure 23B). Control mice received P-particles containing Al(OH)3 or hydrogel without antigen. Shown are the mean ODs of the groups.

[0144] Antibody Avidity Evaluation of individual immune sera for the avidity of anti-GII.4 IgG antibodies (Figure 24) showed that immunization with P-particles alone induced antibodies with significantly lower avidity (avidity index <10%) compared to antibodies induced by the combination of P-particles with the continuous hybrid / composite hydrogel R217-0.2 or the two-phase hybrid / composite hydrogel #1R217-0.2 / R40-0.2A hydrogel (25 wt% antigen in R217-0.2) (avidity index >60%). Approximately the same level was induced by P-particles formulated with Al(OH)3. This analysis shows that R217-0.2 improves antibody affinity.

[0145] Figure 24 shows the avidity of serum IgG antibodies in mice immunized with two 10 μg doses of P-particles (pp) alone or formulated with R217-0.2 hydrogel (Figure 24A), or one 20 μg dose of P-particles alone or formulated with R217-0.2 / R40-20 hydrogel (Figure 24B). Control mice received P-particles containing Al(OH)3 or antigen-free hydrogel. The mean avidity index (%) of the groups is shown.

[0146] Cross-reactive serum IgG reaction Serum antibody cross-reactivity was measured against four heterologous NoV VLPs from genogroup II (GII.4 NO, GII.4 Sydney, GII.12, and GII.17). No cross-reactive IgG antibodies were detected after immunization with P-particles alone, whereas formulations containing R217-0.2 hydrogel or Al(OH)3 yielded antibodies with fairly broad cross-reactivity (Figure 25). The results suggest that the increased cross-reactive antibody response is due to R217-0.2 but not R40-0.2.

[0147] Figure 25 shows cross-reactive serum IgG responses to heterologous NoV VLPs in mice immunized with two 10 μg doses of P-particles (pp) alone or formulated with sequential hybrid / composite R217-0.2 hydrogel (Figure 25A), or one 20 μg dose of P-particles alone or formulated with two-phase hybrid / composite #1 R217-0.2 / R40-0.2 hydrogel (Figure 25B). Control mice received P-particles containing Al(OH) or antigen-free hydrogel. Shown are the mean ODs for the groups.

[0148] neutralizing antibody The neutralizing capacity of the induced antibodies was examined by measuring their blocking activity against homologous GII.4 VLPs using a PGM-based blocking assay. Experimental groups receiving a 10 μg dose of the P particle formulation or a 20 μg dose of the two-phase hybrid / complex #1 formulated with R217-0.2 / R40-0.2A (25 wt% antigen in R217-0.2) produced antibodies with detectable blocking ability, and co-administration of the antigen with sequential hybrid / complex R217-0.2 hydrogel or Al(OH)3 (Figure 26) significantly increased activity. Only P-particles formulated with R217-0.2 hydrogel or Al(OH)3 induced antibodies capable of blocking VLP binding by more than 50%. Based on the analysis, the greatest increase in blocking antibodies came from the formulation containing R217-0.2 and Al(OH)3.

[0149] Figure 26 shows homologous blocking of GII.4 VLP binding to the HBGA receptor by serum antibodies from mice immunized with two 10 μg doses of P-particles (pp) alone or formulated with R217-0.2 hydrogel (Figure 26A) or one 20 μg dose of P-particles alone or formulated with R217-0.2 / R40-0.2 hydrogel (Figure 26B). Control mice received P-particles containing Al(OH)3 or antigen-free hydrogel. Shown are the mean blocking index (%) of the groups.

[0150] mucosal antibodies Mucosal antibody analysis showed that immunization with P-particles alone resulted in very low (or negative) levels of fecal antibodies, whereas formulation of the antigen with sequential hybrid / composite R217-0.2 hydrogel or Al(OH)3 increased the magnitude of mucosal antibodies (Figure 27).

[0151] Figure 27 shows the endpoint titration of fecal IgG antibodies in mice immunized with two 10 μg doses of P-particles (pp) alone or formulated with R217-0.2 hydrogel (Figure 27A), or one 20 μg dose of P-particles alone or formulated with the two-phase hybrid / complex R217-0.2 / R40-0.2 hydrogel (Figure 27B). Control mice received P-particles containing Al(OH)3 or antigen-free hydrogel. Shown is the mean blocking index (%) for the group. Shown is the mean OD for the group.

[0152] conclusion The continuous hybrid / composite R217-0.2 hydrogel functions as an adjuvant to increase the magnitude of serum and mucosal immune responses and to improve the quality and function of the immune response in terms of antibody avidity, cross-reactivity, and neutralizing antibodies.

[0153] The observed adjuvant effect of R217-0.2 is comparable to that of Al(OH)3 hydrogels. Proteins released from the hydrogel resemble the original P-particles by DLS. Furthermore, DLS shows that P-particles aggregate at temperatures above 55°C.

[0154] It was also shown that 16 nm particles could be released from the hydrogel after storage at room temperature and 37°C, but not after storage at 50°C, and the concentration of released particles appeared to decrease over time. Compared to storage in solution, the present hydrogel appeared to have an increased proportion of 15 nm particles at 37°C after storage for more than 5 weeks, suggesting stabilization of P-particles in the hydrogel.

Claims

1. A hydrogel material comprising a first hydrogel, the first hydrogel comprising: - 0.15 wt% to 21 wt% of a functionalized triblock molecule having the structure of formula (1): 【Chemistry 1】 （１） wherein n is 4 to 680 and m is 1 to 10, based on the total weight of the first hydrogel; - 0.85 to 4.0 wt. % silica, based on the total weight of the first hydrogel; and - 75 to 99% by weight of an aqueous liquid based on the total weight of the first hydrogel; The -Si-OH groups of the silica are linked to the -Si-(O) groups of the functionalized triblock molecule of formula (1). ３ - groups and forms -Si-O-Si- bonds.

2. the functionalized triblock molecule of formula (1) and silica form colloidal particles; 10. The hydrogel material of claim 1, wherein the network of particles forms a continuous solid phase within which an aqueous liquid phase is uniformly distributed, and wherein the solid phase and the aqueous liquid phase are within a single hydrogel entity.

3. The method further comprises the step of: - 0.15 to 21 wt. % of said functionalized triblock molecule of formula (1), based on the total weight of said second hydrogel; - 0.85 to 4.0% by weight of silica, based on the total weight of said second hydrogel; - 75 to 99% by weight of an aqueous liquid based on the total weight of the second hydrogel, Here, the -Si-OH groups of the silica are substituted with -Si-(O) groups of the functionalized triblock molecule of formula (1) of the second hydrogel. ３ - group and forms an -Si-O-Si- bond, and wherein the second hydrogel is in the form of uniformly distributed particles dispersed within the first hydrogel, the first hydrogel forming a continuous phase; 3. The hydrogel material of claim 1, wherein the total amount of silica and the functionalized triblock molecule having formula (1) in the second hydrogel is greater than the total amount of silica and the functionalized triblock molecule having formula (1) in the first hydrogel.

4. the functionalized triblock molecule of formula (1) and a first portion of an aqueous liquid are in the form of particles obtained by decomposing a hydrogel of the functionalized triblock molecule of formula (1) and an aqueous liquid into particles dispersed in a silica sol obtained by mixing with a second portion of the aqueous liquid; and The —Si—OH groups of the silica are replaced by —Si—(O) groups of the functionalized triblock molecule of formula (1). ３ 2. The hydrogel material of claim 1, wherein the - group forms an -Si-O-Si- bond with the - group, thus forming a hydrogel material.

5. The hydrogel material according to claim 1 , wherein the silica is alkoxysilane-derived silica.

6. The hydrogel material described in Claim 5, wherein the silica is silica derived from tetraethoxysilane.

7. The aqueous liquid of the first hydrogel and the second hydrogel is 4. The hydrogel material of claim 3, wherein the hydrogel is independently selected from water, a mixture of water and ethanol, and a biologically compatible buffer.

8. 8. The hydrogel material of claim 7, wherein the aqueous liquid is a mixture of water and ethanol, containing 50 to 95% by weight of water, the remainder being ethanol.

9. 9. The hydrogel material of claim 2, 5, 6, 7 or 8, further comprising at least one encapsulated biologically active agent in the first hydrogel.

10. 9. The hydrogel material of claim 3, 7 or 8, further comprising at least one encapsulated biologically active agent in at least one of the first hydrogel and the second hydrogel.

11. 9. The hydrogel material of claim 4, further comprising at least one biologically active agent encapsulated in the hydrogel of the functionalized triblock molecule of formula (1).

12. 12. The hydrogel material of claim 9, wherein the biologically active agent is selected from the group consisting of immunomodulatory agents and therapeutically active agents.

13. 13. Use of the hydrogel material of any one of claims 1 to 12 for the protective encapsulation of a biologically active agent.

14. 14. The use of claim 13, wherein the biologically active agent is encapsulated to ensure thermal stability.

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