Lymphatic-draining polysaccharide cross-linked colloidal particle-based functional drug having reduced immunogenicity

Abstract

The present invention relates to polysaccharide cross-linked colloidal particles and various uses thereof. The polysaccharide cross-linked colloidal particles are water-soluble polysaccharide cross-linked colloidal particles in which -OH functional groups of cross-linking target polysaccharides dispersed in an aqueous solvent are modified with a cross-linker and the surface charge of the particles is controlled within the range of -20 mV to 0 mV through -COOH functional groups exposed on the surface, and which thus have reduced or minimized immunogenicity to immune cells compared to the cross-linking target polysaccharides, and can be excreted from the body without being hydrolyzed by enzymes. The polysaccharide cross-linked colloidal particles are characterized in that, in an aqueous solvent, (i) an -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide, is modified with a first cross-linker having an epoxide group, and a polysaccharide cross-linked particle is formed (a) directly between the functional group modified with the first cross-linker and a spatially adjacent -OH functional group, and / or (b) by cross-linking two spatially adjacent functional groups, modified with the first cross-linker, intramolecularly and / or intermolecularly via a second cross-linker having two or more amine groups (-NH2), and (ii) the number of basic amine groups derived from the cross-linkers exposed on the surface is controlled through modification with -COOH functional groups.

Description

Technical Field [1] The present invention relates to a functional drug based on crosslinked polysaccharide colloidal particles capable of being drained into lymphatic vessels and having reduced immunogenicity, and to various uses thereof. [2] Specifically, the present invention relates to a water-soluble crosslinked polysaccharide colloidal particle and various uses thereof, wherein modification of the -OH functional groups of a polysaccharide to be cross-linked dispersed in an aqueous solvent with a crosslinking agent, together with adjustment of the surface charge of the particle within a range of -20 mV to 0 mV through surface-exposed -COOH functional groups, reduces or minimizes immunogenicity toward immune cells relative to the polysaccharide to be cross-linked and allows the particle to be eliminated from the body without being hydrolyzed by enzymes in the body. The crosslinked polysaccharide colloidal particle is characterized in that, in an aqueous solvent, (i) the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide are modified with a first crosslinking agent having an epoxide group, and a crosslinked polysaccharide particle is formed by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2), and (ii) the number of surface-exposed crosslinker-derived basic amino groups is adjusted through modification into -COOH functional groups. Background Art [3] Polysaccharides, together with other biomolecules such as proteins and nucleotides, are essential components and perform numerous functions in biological systems, including intercellular communication, adhesion, and molecular recognition in the immune system. [4] Dextran is a complex branched polysaccharide extracted from bacteria and may be recognized as a foreign antigen by the immune system when introduced into the body. Dextran (MW 40 kDa) has inhibitory effects on platelet aggregation and coagulation factors and is used as a blood plasma volume expander, but adverse drug reactions such as pain, inflammation, and edema may occur. [5] Macrophages express various polysaccharide-binding surface receptors to recognize foreign pathogens or damaged cellular debris. These receptors interact with polysaccharide components, such as P-glucan, mannose, fucose, and N-acetylglucosamine, to activate signaling pathways that induce immune responses or regulate inflammatory responses. In this regard, TLR4, mannose receptor (CD206), Dectin-1, complement receptor 3 (CR3), and scavenger receptor (SR) have been identified as representative polysaccharide-binding receptors. [6] Ligands for Toll-like receptor 4 (TLR4) include P-glucan (e.g., fungal cell walls), bacterial lipopolysaccharide (LPS), and ginseng polysaccharides. TLR4 recognizes ligands by forming a complex with CD14 or MD-2 and activates MAPK (ERK1 / 2, JNK, p38) and NF-kB through a MyD88-dependent pathway, thereby promoting the secretion of pro-inflammatory cytokines such as TNF-a and IL-6. Through a synergistic effect with IFN-y, TLR4 also increases the activity of the transcription factor AP-1, thereby amplifying the secretion of NO and cytokines. [7] Ligands for the mannose receptor (CD206) include mannose, fucose, and N-acetylglucosamine. The receptor binds to saccharides in a Ca2+-dependent manner through a C-type lectin domain (CRD4) and is primarily involved in endocytosis and antigen presentation. Although its direct signaling pathways have not yet been fully elucidated, its saccharide-binding structure has been analyzed in detail. [8] Ligands for Dectin-1 are fungal polysaccharides such as P-1,3-glucan. Dectin-1 activates Syk kinase and CARD9 to induce the secretion of IL-1P, IL-6, and ROS through the NF-kB pathway and promotes the secretion of TNF-a and IL-12 through cooperation with TLR2. For example, polysaccharides from Inonotus obliquus have been reported to bind to Dectin-1 and induce activation of the tumor-killing activity of macrophages. [9] Ligands for complement receptor 3 (CR3; CD11b / CD18) are complement-opsonized pathogens and P-glucan. CR3 activates the MAPK / AP-1 pathway in cooperation with TLR4 or CD14 and inhibits NO secretion while promoting phagocytosis in LPS-activated macrophages.

[10] Ligands for scavenger receptors (SR) are sulfated polysaccharides, such as dextran sulfate. These receptors induce the NF-kB and PI3K / AKT pathways to regulate the balance between pro-inflammatory and anti-inflammatory responses, and increased SR expression in BCG-activated macrophages enhances polysaccharide-binding capacity.

[11] Polysaccharide-binding receptors on the surface of macrophages fine-tune immune responses through mutual cooperation and signaling crosstalk rather than acting individually. Macrophages use various polysaccharide-binding receptors to eliminate pathogens and regulate inflammation, and cooperation among these receptors plays an important role in precisely fine-tuning immune responses.

[12] NF-kB is a transcription factor that plays an important role in regulating immune responses, inflammation, and cell survival. NF-kB increases the levels of signaling molecules involved in inflammation and immune responses, as well as important substances involved in cell proliferation, apoptosis, and the cell cycle.

[13] The canonical NF-kB activation pathway forms part of a signaling system essential for innate immunity and involves the translocation of cytoplasmic NF-kB into the nucleus and its activation through IKKp—dependent IkB degradation.

[14] When a cell is stimulated externally through pathogen-associated molecular patterns (PAMPs), TNF receptors (TNFR) or Toll-like receptors (TLR), IL-1 receptors (IL-1R), or the like, the IKK complex causes IkB, which binds to an NF-kB dimer and inhibits its activation, to undergo phosphorylation and polyubiquitination followed by degradation in the proteasome, thereby releasing the NF-kB dimer from IkB and activating the dimer. The NF-kB dimer released from IkB, usually a p50-p65 dimer, passes through the nuclear membrane, enters the nucleus, and binds to DNA to activate gene transcription. The canonical NF-kB activation pathway is known to activate the transcription of genes encoding various chemokines, cytokines, ICAM-1, VCAM-1, endothelial leukocyte adhesion molecule-1 (ELAM), secondary inflammatory mediator enzymes, and enzymes that inhibit apoptosis.

[15] Meanwhile, B cells have surface receptors known as B-cell receptors (BCRs) that can bind to specific antigens. Polysaccharides are often T-cell-independent antigens. In a T-cell-independent antigen response, an antigen may induce activation by cross-linking multiple BCRs on the surface of a B cell.

[16] The NF-kB signaling pathway plays an essential role in B-cell survival, proliferation, and regulation of immune responses and is closely linked to BCR signaling. This pathway is activated through a canonical IKK-dependent pathway and an alternative (IKK-independent) pathway, and dysregulation of these pathways is associated with various pathological conditions, such as lymphoma and autoimmune diseases. NF-kB contributes to the survival and development of pre-B cells by increasing the expression of anti-apoptotic proteins (Bcl-2, Bcl-xL). Through interaction with the AKT / FOXO1 pathway, NF-kB promotes the expression of MYC and cyclins, thereby inducing cell division.

[17] Chemical crosslinking of polysaccharides forms a three-dimensional network structure through chemical reactions. Physical properties, such as the size, surface charge, and porosity of particles synthesized by chemically crosslinking polysaccharides, are factors that significantly affect the biodistribution and residence time in the body of drugs based on chemically crosslinked polysaccharides. Accordingly, even when synthesis conditions, such as reaction temperature, reaction time, pH, and reactant concentration, are precisely controlled, subtle differences may arise in the degree of crosslinking, molecular weight distribution, and arrangement of functional groups. Such structural heterogeneity causes changes in particle solubility, physical stability, and interactions with biomolecules, such as serum proteins and cell receptors, such that even nanoparticles based on the same dextran may exhibit different pharmacokinetic behavior in the body, including metabolism, excretion, and residence time in the body.

[18] In addition, because polysaccharides have polymeric and complex structures, their metabolic and excretory pathways differ among species. Thus, the metabolic stability or elimination rate of drugs based on chemically crosslinked polysaccharides in animal models may likewise not correspond to metabolic mechanisms in humans. This may result in unexpected drug accumulation or the formation of toxic metabolites.

[19] Furthermore, drugs based on chemically crosslinked polysaccharides may be recognized differently by the immune systems of different species. In particular, expression patterns of polysaccharide-recognizing receptors, such as lectins and scavenger receptors, differ greatly among rodents, primates, and humans. For example, the same crosslinked polysaccharide nanoparticles may be rapidly eliminated by the reticuloendothelial system (RES) in mice, but may persist longer in humans and induce unexpected complement activation or cytokine secretion.

[20] When a foreign substance is introduced into the body, it is recognized by innate immune receptors (PRRs), B-cell receptors (BCRs), and the like, thereby triggering an immune response. In particular, in B cells, BCRs cluster in a specific spatial region to determine signal strength and duration. If abnormal immune stimulation consequently occurs, B-cell proliferation, differentiation, and antibody production are promoted, creating a risk of allergy, inflammation, hypersensitivity reactions, and the like. Existing drug delivery systems have limitations in fully addressing adverse effects arising from such immunogenicity, such as rapid carrier elimination and reduced therapeutic efficacy.

[21] Conventional polysaccharide-based drugs or nanoparticles have also frequently elicited recognition through receptors on immune cells, resulting in rapid elimination from the body, adverse effects, and reduced therapeutic effects. In particular, unpredictability arising from differences in immune receptor expression and metabolic pathways between animal models and humans has emerged as a problem. Disclosure of the Invention Technical Problem

[22] The present invention seeks to provide non-immunogenic crosslinked polysaccharide colloidal particles capable of addressing the aforementioned unpredictability, associated with drugs based on chemically crosslinked polysaccharides, of pharmacokinetic behavior in the body, including metabolism, excretion, and residence time in the body, and of biological activity resulting from interactions with biomolecules, such as polysaccharide-recognizing receptors and BCRs.

[23] Turning to advantage the problem that binding to surface receptors on immune cells may differ among species even when the same polysaccharide is chemically crosslinked to the same degree of crosslinking, the present invention seeks to provide a platform of non-immunogenic crosslinked polysaccharide colloidal particles having a compact, spherical, three-dimensional network structure in which interactions with surface receptors on immune cells are minimized and immune activation is suppressed even upon repeat administration despite subtle differences. This is achieved, even when various polysaccharide polymers are used as polysaccharides to be cross-linked, by selectively modifying the -OH functional groups of monosaccharides with an epoxide-based first crosslinking agent and precisely controlling chemical crosslinking and crosslinking conditions, such as the type and amount of crosslinking agent, reaction temperature, reaction time, pH, and reactant concentration.

[24] In addition, the present invention seeks to provide a platform technology based on non-immunogenic crosslinked polysaccharide colloidal particles that can effectively overcome immune responses generally elicited by polysaccharide-based drugs or nanoparticles administered into the body, the resulting adverse reactions, such as allergy, inflammation, hypersensitivity reactions, and edema, and reduced efficacy of therapeutic agents or contrast agents.

[25] Furthermore, the present invention seeks to provide crosslinked polysaccharide colloidal particles that have reduced or minimized immunogenicity toward immune cells relative to the polysaccharide to be cross-linked, which may cause adverse immune reactions when administered into the body, and that are eliminated from the body without being hydrolyzed by enzymes in the body.

[26] In addition, the present invention seeks to provide crosslinked polysaccharide colloidal particles that (a) are filtered through renal capillaries but do not pass through the walls of normal capillaries and optionally (b) are drained exclusively into peripheral lymphatic vessels.

[27] A first aspect of the present invention provides a non-immunogenic, water-soluble crosslinked polysaccharide colloidal particle in which -OH functional groups of a polysaccharide to be cross-linked, dispersed in an aqueous solvent, are modified with a crosslinking agent and the surface charge of the particle is adjusted to within a range of -20 mV to 0 mV through surface-exposed -COOH functional groups, thereby reducing or minimizing immunogenicity toward immune cells compared with the polysaccharide to be cross-linked and allowing the particle to be eliminated from the body without being hydrolyzed by enzymes in the body,

[28] wherein the crosslinked polysaccharide colloidal particle is formed in an aqueous solvent by: (i) modifying -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, and forming a crosslinked polysaccharide particle through intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2); and (ii) adjusting the number of surface-exposed crosslinker-derived basic amino groups through modification into -COOH functional groups.

[29] In the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention, 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide may be modified with a crosslinking agent such that the particle is not hydrolyzed by enzymes in the body and has reduced or minimized immunogenicity toward immune cells.

[30] In addition, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be such that at least one -OH functional group in at least one of two consecutive monosaccharides within a polysaccharide chain is predominantly modified with a crosslinking agent, whereby the particle is not hydrolyzed by enzymes in the body and has reduced or minimized immunogenicity toward immune cells.

[31] When the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention has a hydrodynamic diameter of 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, the particle may (a) be filtered through renal capillaries but not pass through the walls of normal capillaries and optionally (b) be drained exclusively into peripheral lymphatic vessels.

[32]

[33] A second aspect of the present invention provides a pharmaceutical composition characterized in that the non-immunogenic crosslinked polysaccharide colloidal particle of the first aspect is used as a drug carrier for early diagnosis of an inflammatory disease or as a platform for lesion-targeted delivery.

[34] A third aspect of the present invention provides a pharmaceutical composition characterized in that the non-immunogenic crosslinked polysaccharide colloidal particle of the first aspect is used as a contrast agent for quantitative metrics in an AI-based image analysis system.

[35] A fourth aspect of the present invention provides a diagnostic composition characterized in that the non-immunogenic crosslinked polysaccharide colloidal particle of the first aspect is used as a contrast agent capable of differentiating inflammatory lesions or determining their stage.

[36]

[37] The present invention is described below.

[38] Conventionally, administration of a foreign substance into the body gives rise to the problem that the substance is recognized by surface receptors on immune cells, such as innate immune receptors (PRRs) and B-cell receptors (BCRs), thereby eliciting immune responses and adverse effects, such as allergy, inflammation, hypersensitivity reactions, and edema. Such immune responses may lead to rapid clearance of a drug carrier and reduced efficacy of a therapeutic agent or contrast agent. Accordingly, the present invention provides a new platform technology that overcomes these problems and minimizes recognition by immune cells in the body while ensuring stable biodistribution and elimination from the body.

[39] To address the problem of adverse immune reactions, the present invention provides a platform of non-immunogenic crosslinked polysaccharide colloidal particles having a compact, spherical, three-dimensional network structure that does not elicit immune stimulation through interactions with surface receptors on immune cells. This is achieved, even when various polysaccharide polymers are used as polysaccharides to be cross-linked, by selectively modifying the -OH functional groups of monosaccharides with an epoxide-based first crosslinking agent and precisely controlling chemical crosslinking and crosslinking conditions, such as the type and amount of crosslinking agent, reaction temperature, reaction time, pH, and reactant concentration.

[40] In particular, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, which have a compact, spherical, three-dimensional network structure, are designed / synthesized to bind weakly to surface receptors on immune cells, such as innate immune receptors (PRRs, pattern recognition receptors) and BCRs, during distribution in the body, such that they do not act as an external stimulus that turns on the immune response switch (an immune cell activation signal) and, consequently, do not induce B-cell activation or antibody production.

[41] The present invention is based on the premise that chemical crosslinking of various polysaccharide polymers, such as dextran, gives rise to subtle differences in the degree of crosslinking, molecular weight distribution, and arrangement of functional groups within the three-dimensional network structure. However, a key feature of the present invention is that, despite such structural heterogeneity, the surface of the compact, spherical, three-dimensional network structure formed by selectively modifying the -OH functional groups of monosaccharides, which constitute the repeating structure of the polysaccharide to be crosslinked, with an epoxide-based first crosslinking agent and chemically crosslinking the 8 polysaccharide is optimized so as not to interact with pattern recognition receptors (PRRs, e.g., TLRs and C-type lectin receptors) or B-cell receptors (BCRs) on immune cells.

[42] Specifically, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention not only provides a compact, spherical, three-dimensional network structure in which the polysaccharide is chemically crosslinked by selectively modifying the -OH functional groups of monosaccharides with an epoxide-based first crosslinking agent, preferably a three-dimensional network structure in which 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent, but also has a surface charge adjusted to a range of -20 mV to 0 mV by controlling the number of crosslinker-derived amino groups exposed on the particle surface and modifying some or all of the amino groups into carboxylic acid groups (-COOH). The particle is thereby characterized by minimized recognition by surface receptors on immune cells and suppressed immune activation even upon repeat administration.

[43] Inhibition of binding to immune cells: When a polysaccharide polymer is crosslinked at the -OH functional groups of its monosaccharides to form compact, nearly spherical nanoparticles according to the present invention, the rotational freedom of chemical bonds is drastically reduced, making it difficult to provide a three-dimensional structure capable of binding to pattern recognition receptors (PRRs), BCRs, or the antigen-binding sites of antibodies through a combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions. Accordingly, binding strength to PRRs and BCRs is markedly reduced, thereby blocking signaling that induces immune responses.

[44] Stability upon repeat administration: Such structural optimization prevents immune activation and antibody production (anti-drug antibodies, ADAs) even upon repeat administration, thereby ensuring safety during long-term treatment and use of diagnostic agents, such as contrast agents.

[45] Accordingly, the crosslinked polysaccharide colloidal particles of the present invention effectively block immunogenic sites of polysaccharides that may act as foreign antigens, thereby suppressing activation of immune cells, such as macrophages and dendritic cells, and induction of antibody production. In particular, in B cells, abnormal immune signaling caused by BCR clustering is blocked, greatly reducing the risk of allergy and hypersensitivity reactions.

[46] In addition, in the crosslinked polysaccharide colloidal particles of the present invention, the robust three-dimensional network formed through chemical crosslinking by selectively modifying the -OH functional groups of monosaccharides with an epoxide-based first crosslinking agent provides resistance to hydrolysis by enzymes in the body. Achieving an optimal particle size by controlling the degree of crosslinking and appropriately adjusting the surface charge by controlling the number of crosslinker-derived amino groups exposed on the particle surface enable stable elimination from the body through normal metabolic and elimination pathways, such as those involving the kidneys and the lymphatic system.

[47] Accordingly, taking into account the complexity of polysaccharide structures and speciesdependent differences in recognition by the immune system, the crosslinked polysaccharide colloidal particles of the present invention are designed to compensate for differences in metabolic stability and elimination rate between animal models and humans. This reduces the possibility of unexpected drug accumulation or formation of toxic metabolites in human clinical settings.

[48] In summary, the platform technology based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention minimizes structural heterogeneity that may arise during formation of a three-dimensional network through polysaccharide crosslinking by providing a compact, spherical, three-dimensional network structure in which the polysaccharide is chemically crosslinked by selectively modifying the -OH functional groups of monosaccharides with an epoxide-based first crosslinking agent, preferably a threedimensional network structure in which 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent, and inhibits degradation by enzymes in the body. In addition, surface modification technology minimizes interactions with immune cells and enables stable elimination from the body. Furthermore, by compensating for interspecies differences in immune recognition and variability in metabolic pathways, the technology can effectively prevent immune responses and adverse effects caused by foreign substances.

[49] Consequently, the crosslinked polysaccharide colloidal particles of the present invention may be applied to early diagnosis of inflammatory diseases, lesion-targeted delivery, and development of safe drug delivery systems. In particular, by minimizing adverse effects associated with immune responses in the body, such as allergy, inflammation, hypersensitivity 10 reactions, and edema, the particles can effectively address the problem of reduced therapeutic efficacy observed with existing systems.

[50] The present invention was completed by confirming from MRI images (FIG. 18) that the T1 MRI contrast agent based on the crosslinked polysaccharide colloidal particles of the present invention is transported as far as the central lymphatic vessels without being retained through interactions with immune cells in lymph nodes where various immune cells gather (Example 5-7), and by elucidating for the first time the associated physicochemical and structural characteristics achieved through the method for preparing the crosslinked polysaccharide colloidal particles of the present invention.

[51] In addition, preclinical animal studies confirmed that no toxicity occurred even upon repeat administration, provided that the dose was adjusted to prevent gelation or aggregation at the administration site (Table 4 and the repeat-dose NOAEL in Table 7).

[52] Furthermore, clinical trials and animal studies showed that the crosslinked polysaccharide colloidal particles of the present invention not only do not elicit immune responses, such as adverse immune reactions, in the immune system encountered following administration into the body, during drainage into lymphatic vessels and circulation through the vascular system, and subsequent excretion in urine through the kidneys, but also are not hydrolyzed by glycoside hydrolases distributed in the body, thereby preventing exposure of potentially immunogenic sites.

[53] The crosslinked polysaccharide colloidal particle according to the present invention is characterized in that (i) it is an amorphous, water-soluble colloidal particle formed in an aqueous solvent by modifying the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, followed by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2), and (ii) the number of surface-exposed crosslinker-derived basic amino groups is adjusted through modification into -COOH functional groups.

[54] Here, the crosslinked polysaccharide colloidal particle according to the present invention, in which the -OH functional groups of a polysaccharide to be cross-linked dispersed in an aqueous solvent are modified with a crosslinking agent and the surface charge of the particle is adjusted within a range of -20 mV to 0 mV through surface-exposed -COOH functional groups, may have a hydrodynamic diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and still more preferably 6 nm or less.

[55] The crosslinked polysaccharide colloidal particles according to the present invention, in which the -OH functional groups of a polysaccharide to be cross-linked dispersed in an aqueous solvent are modified with a crosslinking agent,

[56] when 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent, the hydrodynamic diameter is 2 to 10 nm, and the surface charge of the particles is adjusted within a range of -20 mV to 0 mV through surface-exposed -COOH functional groups,

[57] were found to (a) be filtered through renal capillaries but not pass through the walls of normal capillaries, (b) be drained exclusively into peripheral lymphatic vessels without venous contamination, depending on the administration site, and (c) not only have reduced or minimized immunogenicity toward immune cells relative to the polysaccharide to be crosslinked, but also be eliminated from the body without exposure of potentially immunogenic saccharide-based sites because the particles are not hydrolyzed by enzymes in the body (Examples 3, 4, 5, and 6).

[58] In addition, the present inventors found that, when complex branched dextran or a derivative thereof is crosslinked with a crosslinking agent at the -OH functional groups of its glucose building blocks in an aqueous solution, one to three dextran or dextran derivative polymer molecules form spherical nanoparticles through intramolecular and / or intermolecular crosslinking, and that these crosslinked dextran-based nanoparticles exhibit compressibility and excellent colloidal stability without aggregation. The inventors also confirmed that the crosslinker-derived functional groups exposed on the surfaces of the crosslinked dextran-based nanoparticles can be used to modify the surfaces of functional nanoparticles for introduction into the body and to modify drugs (Examples 1 and 2). By adjusting the type and / or degree of substitution of substituents replacing the crosslinker-derived functional groups exposed on the surfaces of the crosslinked dextran-based nanoparticles, the surface charge could be freely controlled (Examples 1-5 and 1-6). In addition, the number of functional molecules, such as drug molecules, bound to the crosslinked dextran-based nanoparticles could be controlled by adjusting the reaction ratio with the drug (Example 1-7). It was also confirmed that, because the hydrodynamic diameter of the crosslinked dextran-based nanoparticles increases as the molecular weight of the dextran used to synthesize the nanoparticles for use as a modifying agent increases, the hydrodynamic diameter of a complex for injection into the body that is modified with the crosslinked dextran-based nanoparticles can be controlled.

[59] In addition, the squeezability of crosslinked dextran-based nanoparticles (cross-linked dextran nanoparticles, C-DNPs), relative to inorganic nanoparticles, was confirmed to be suitable for the in vivo biological environment through which the particles pass, namely, biological microstructures in the body. The squeezability of iron oxide nanoparticles coated with C-DNPs, and their resulting diffusion and mobility within biological tissues, were also confirmed. Furthermore, synthesis and analysis of iron oxide nanoparticles coated with C-DNPs of various molecular weights revealed that their size can be controlled because the hydrodynamic size increases as the molecular weight of the dextran used to synthesize the C-DNPs increases; that the surface charge of the coated iron oxide nanoparticles can be controlled by adjusting the surface charge of the C-DNPs; that C-DNP-coated iron oxide nanoparticles exhibit excellent colloidal stability, remaining dispersed for a long time without precipitation caused by aggregation under various physiological conditions involving salts and pH; that the use of compressible C-DNPs having surface-exposed crosslinker-derived functional groups capable of forming coordination bonds enables iron oxide nanoparticles to be coated using onetwentieth the amount required when using non-crosslinked dextran while securing excellent colloidal stability; that the anti-opsonization effect of C-DNP-coated iron oxide nanoparticles can be controlled by adjusting the surface charge of the nanoparticles; and that the pharmacokinetic behavior of C-DNP-coated iron oxide nanoparticles can be controlled by adjusting the size and surface charge of the C-DNPs.

[60] As described above, the crosslinked polysaccharide colloidal particles of the present invention were experimentally demonstrated to constitute a non-immunogenic platform capable of effectively avoiding immune-mediated inflammatory responses, such as pain, erythema, and edema, commonly observed with conventional drugs based on foreign substances. When crosslinked dextran (C-DNP) was intravenously administered to rats at the same doses (125 and 250 mg / kg) as dextran T-10 before crosslinking, acute edema in the face and limbs was observed only in the dextran-treated group, whereas the polysaccharide colloidal particles of the present invention having a high degree of crosslinking (NEMO-103, degree of crosslinking >60%) did not cause adverse effects under the same conditions (see Example 2-1 and FIG. 2). This suggests that crosslinking masks immune recognition structures on the polysaccharide surface, thereby inhibiting interactions with immune cell receptors.

[61] In addition, evaluation of the activation of NF-kB, a key signaling factor in inflammation and immune responses, confirmed that the crosslinked particles of the present invention do not significantly stimulate the NF-kB transcriptional pathway in macrophages despite stimulation with PAMPs (FIGS. 4 and 6). This demonstrates that the particles according to the present invention possess biocompatibility that allows their stable use in the body without stimulating the immune system.

[62] Furthermore, in Example 2, in which the crosslinked polysaccharide colloids of the present invention were used as drug carriers, drug conjugates were prepared with small-molecule ligands for cancer immunotherapy, such as TLR 7 / 8 agonists, uniformly distributed on their surfaces. In vitro and in vivo experiments confirmed that these conjugates effectively induce intracellular signaling and immune cell activation by specifically binding to cell membrane receptors (see FIGS. 4, 5, 6, and 7). These results show that the present invention can simultaneously impart immune evasion properties and drug delivery capability to polysaccharide-based biomaterials through a highly controlled crosslinking reaction.

[63] In Example 4, NEMO-103, a crosslinked dextran-based T1 contrast agent, was synthesized, and its physicochemical properties and imaging efficacy in a Phase 1 / 2a clinical trial were evaluated. NEMO-103 was used in MR arthrography of the shoulder and provided image quality equivalent to or better than that of the control (a GBCA-based contrast agent), as assessed by CNR, distension, and Turing test criteria. The results also suggested the possibility of extending the imaging window by improving the resistance of the contrast agent to resorption. In addition, a 28-day evaluation of colloidal stability under physiological conditions of pH 5-9 and NaCl concentrations up to 1,000 mM confirmed that the contrast agent of the present invention remained stable without aggregation.

[64] For the purpose of imaging lymphatic vessels without venous contamination, Example 5 evaluated the applicability of INV-001, an iron-based T1 contrast agent, to magnetic resonance lymphangiography (MRL) in various animals. INV-001 has a structure in which iron ions are coordinated to crosslinked dextran colloids. Its hydrodynamic size (~4 nm) is smaller than the renal filtration limit, such that, upon intradermal injection, it was selectively absorbed via the lymphatic system and eliminated without being absorbed into the venous system. Following intradermal injection of INV-001 into both hind limbs of Sprague-Dawley rats (250-300 g), imaging using a 3D TOF sequence on a 9.4 T MRI system showed no venous contamination in the INV-001-treated group, in contrast to Gd-DOTA. In particular, INV-001 showed maximum enhancement of the popliteal lymph nodes and lymphatic vessels between 16 and 32 minutes after injection, and clear lymphatic images could be obtained even within a dose range of 0.450.75 pmol without enhancement of the interstitial space or lymph node enlargement.

[65] In Example 5, conducted in healthy beagle dogs, the optimal concentration and dose of INV-001 for MRL were determined. INV-001 is a nanoparticulate contrast agent having a dextran core-iron oxide shell structure. When administered intradermally at a concentration of 15 mM and doses of 0.056 and 0.112 mg Fe / kg, respectively, it enabled selective imaging of only lymphatic vessels and lymph nodes, with no venous contamination observed. This shows that INV-001 provides lymphatic image quality equivalent to or better than that of conventional GBCA-based contrast agents (typical concentration: 500-1,000 mmol / L; administered dose: 0.1-0.2 mL / kg), even at markedly lower concentrations (0.067 and 0.13 mL / kg). These results contribute to establishing preclinical dosing parameters for INV-001 in MRL and support the potential for clinical translation of lymphatic-selective contrast agent development.

[66] Example 6 shows that INV-001 is a lymphatic system-specific T1 MRI contrast agent that consistently provides specific contrast enhancement of lymph nodes and lymphatic vessels in a Phase 1 clinical trial, with excellent safety based on immune inactivity demonstrated even in tissues densely populated with immune cells by findings including the MTD not being reached, the absence of ADRs and SAEs, and the absence of local inflammatory reactions.

[67] Meanwhile, Example 3 confirmed that the crosslinked dextran-based nanoparticles used in Example 1, which are formed by intramolecular and / or intermolecular crosslinking of one to three dextran or dextran derivative molecules with a crosslinking agent at the -OH functional groups of their glucose building blocks, can be extended to crosslinked polysaccharide colloidal particles formed by intramolecular and / or intermolecular crosslinking of one to three linear or branched polysaccharide molecules or two to thirty cyclic polysaccharide molecules dispersed in an aqueous solvent, using a crosslinking agent at the -OH functional groups of their monosaccharide building blocks.

[68] Specifically, the crosslinked polysaccharide colloidal particle of the present invention is formed in an aqueous solvent by (i) modifying the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, and forming a crosslinked polysaccharide particle by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2), and (ii) adjusting the number of surface-exposed crosslinker-derived basic amino groups through modification into -COOH functional groups. Such a particle can be provided as a crosslinked polysaccharide colloidal particle having a hydrodynamic diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and still more preferably 6 nm or less, and a surface charge adjusted within a range of -20 mV to 0 mV through surface-exposed -COOH functional groups. Example 3 and FIGS. 8 to 10 showed that, following intravenous injection, the crosslinked polysaccharide colloidal particles enter the blood vessels of the nephrons and are excreted in urine through the renal filtration mechanism, as confirmed by MRI signals in the bladder. These observations confirmed that the particles not only have minimized immunogenicity toward immune cells, for example, in terms of phagocytosis by macrophages and recognition by B cells, in the immune systems of the liver / spleen encountered during vascular circulation and subsequent urinary excretion through the kidneys, but also are not hydrolyzed by enzymes in the body.

[69] In summary, when 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent according to the present invention, at least one -OH functional group in at least one of two consecutive monosaccharides within a polysaccharide chain is predominantly modified with a crosslinking agent. As a result, the crosslinked polysaccharide colloidal particle not only has reduced or minimized immunogenicity toward immune cells relative to the polysaccharide to be cross-linked, but also is eliminated from the body without being hydrolyzed by enzymes in the body.

[70] Here, a monosaccharide within the polysaccharide chain may have two to three -OH functional groups, and a monosaccharide modified with a crosslinking agent may have one of its two to three -OH functional groups modified with the crosslinking agent.

[71] As the molecular weight of the polysaccharide to be cross-linked increases, a greater number of monosaccharides modified with a crosslinking agent among the total number of monosaccharides within the polysaccharide chain is desirable in terms of preventing hydrolysis by enzymes in the body and reducing or minimizing immunogenicity toward immune cells relative to the polysaccharide to be cross-linked, thereby allowing elimination from the body without exposure of potentially immunogenic saccharide-based sites.

[72] Meanwhile, the crosslinked polysaccharide colloidal particle of the present invention, in which 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent and the surface charge is adjusted within a range of -20 mV to 0 mV through surface-exposed -COOH functional groups,

[73] may be provided by, in an aqueous solvent, (i) modifying the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, and forming a crosslinked polysaccharide particle by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2), and (ii) adjusting the number of surface-exposed crosslinker-derived basic amino groups through modification into -COOH functional groups.

[74] Here, the crosslinked polysaccharide colloidal particle of the present invention may be designed to have a hydrodynamic diameter of 2 to 20 nm and to have 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide modified with a crosslinking agent by controlling parameters such as the molecular weight of the polysaccharide, the length of the polysaccharide backbone, the type of crosslinking agent used for crosslinking, the amount and addition rate of the crosslinking agent during the synthesis reaction, and additional chemical functional group modification. The intramolecular crosslinking density and / or intermolecular crosslinking density may be adjusted to a high level to minimize adverse immune reactions in the human body.

[75] For example, the present invention may provide a crosslinked polysaccharide colloidal particle having a hydrodynamic diameter of 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, and a surface charge of -20 mV to 0 mV such that the particle (a) is filtered through renal capillaries but does not pass through the walls of normal capillaries and (b) is selectively drained exclusively into peripheral lymphatic vessels without penetrating or being drained into capillaries at the injection site, and (c) has minimized immunogenicity toward immune cells and is not hydrolyzed by enzymes in the body, allowing elimination from the body without exposure of potentially immunogenic saccharide-based sites. The particle exhibits pharmacokinetics (modes of absorption, distribution, metabolism, and excretion) and pharmacodynamics (effects on the body) that are entirely different from those of conventional nanomedicines having different physicochemical properties or crosslinked polysaccharide colloidal particles that do not satisfy all of conditions (a), (b), and (c) above. In addition, the crosslinked polysaccharide colloidal particles according to the present invention have altered biological properties, such as reduced immunogenicity toward immune cells, and therefore may also be used at doses, according to dosing regimens, and / or via routes of administration different from those used for conventional nanomedicines having different physicochemical properties.

[76] Non-limiting examples of crosslinker-derived functional groups or hydrophilic functional groups exposed on the surface of the crosslinked polysaccharide colloidal particle of the present invention include amino groups, carboxyl groups, hydroxyl groups, and / or thiol groups.

[77] The -OH functional groups of monosaccharides constituting the building blocks of the polysaccharide and / or crosslinker-derived functional groups may be modified through various covalent linkages, such as those shown in Table 1 below.

[78] [Table 1] Covalent linkages to polysaccharides (Debele et al., 2016b; Yang et al., 2015b) Functional group Chemical modifications used -OH Esterification with acylating agents Etherification with alkylating agents Oxidation of primary alcohol to COOH Ester linkage to -OH Amide linkage to -NH2 Oxidation of vicinal secondary -OH to aldehydes Hydrazone linkage to - NHNH2 Schiff base linkage to -NH2 -COOH Ester linkage to -OH Amide linkage to -NH2 -NH2 Amide linkage to -COOH

[79] Reactive functional groups, such as amino, thiol, carboxyl, and hydroxyl groups, facilitate not only surface modification but also chemical conjugation with biopharmaceuticals, such as ligands that specifically bind to receptors on particular cells, antibodies or fragments thereof, proteins, peptides, and nucleic acids (DNA, RNA, or fragments thereof, etc.), or with various types of small-molecule drugs.

[80] The crosslinked polysaccharide colloidal particle of the present invention may be bound to nanoparticles, molecules, or metal ions through covalent or coordination bonds via surface-exposed crosslinker-derived amino groups or -COOH functional groups.

[81] Accordingly, the physicochemical properties of the crosslinked polysaccharide colloidal particle according to the present invention can be controlled in diverse and precise ways by variously modifying surface-exposed crosslinker-derived functional groups, such as amino groups, into -COOH functional groups and / or with nanoparticles, molecules, or metal ions, thereby enabling precise control of biodistribution and elimination.

[82] In particular, the surface of the crosslinked polysaccharide colloidal particle of the present invention may be bound through covalent or coordination bonds to functional nanoparticles (e.g., iron oxide nanoparticles, antibodies, proteins having a three-dimensional structure, or nucleic acids), molecules (e.g., small-molecule drugs, hormones, or neurotransmitters), or metal ions via crosslinker-derived amino groups or -COOH functional groups.

[83] Accordingly, the crosslinked polysaccharide colloidal particle according to the present invention may be used as an MRI contrast agent, a drug carrier, and / or a modifying agent. For example, iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) may be coordinated to the crosslinked polysaccharide colloidal particle of the present invention through its surface-exposed crosslinker-derived functional groups, such as amino groups whose number is controlled and / or -COOH functional groups (T1 MRI contrast agent), and the particle may be used to modify the surfaces of functional nanoparticles, hydrophobic drugs, or drugs having a short half-life.

[84] The crosslinked polysaccharide colloidal particle of the present invention conjugated with a small-molecule drug, antibody, or ligand that binds to a cell-surface ligand or receptor expressed at a particular site in the body may be distributed to that site and, after a certain period of time, drained into lymphatic vessels.

[85]

[86] [MR Imaging and Contrast Agents]

[87] When iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), manganese ions (Mn2+), or iron oxide particles are coordinated to surface-exposed crosslinker-derived amino groups or -COOH functional groups of the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention, the particle may be used as an MRI contrast agent that enhances image contrast, a key class of drugs in modern medicine (Example 1 and Examples 3 to 7).

[88] In addition, the MRI contrast agent based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention enables control of biodistribution and elimination by variously controlling and modifying the hydrodynamic diameter, surface charge, and / or surface-exposed crosslinker-derived functional groups to achieve the desired pharmacokinetics (modes of absorption, distribution, metabolism, and excretion). This enables not only diagnosis of deep organs and the creation and expansion of new markets, such as specialized applications for musculoskeletal diseases and lymphatic vascular diseases, but also application to diseases that cannot be diagnosed using existing contrast agents and various diseases that are difficult to visualize using medical diagnostic equipment alone.

[89] When iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) are coordinated to surface-exposed crosslinker-derived amino groups or -COOH functional groups and uniformly distributed on the particle surface, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be used as a T1 MRI contrast agent that increases the relaxation rate of protons in nearby water molecules, thereby increasing signal intensity in T1-weighted MRI images and making the corresponding region appear brighter.

[90] In addition, a drug based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, in which the particles themselves act as a drug or a separate drug is conjugated / loaded, may have iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) coordinated to surface-exposed crosslinker-derived amino groups or -COOH functional groups to impart the function of a T1 MRI contrast agent that produces a bright signal in MRI images. Accordingly, the location of the crosslinked polysaccharide colloidal particle-based drug can be tracked by MRI following injection into the body.

[91] Furthermore, a T1 MRI contrast agent based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention having hydrophobic ligands attached to their surfaces may be used as a T1 MRI contrast agent for determining whether a receptor for a particular hydrophobic ligand is expressed on the surface of a particular cell.

[92] In addition, iron oxide nanoparticles designed to function as T1 or T2 MRI contrast agents may have their surfaces modified with the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, rendering them squeezable so that they can move between structures in the body without aggregation upon injection into the body and perform their intended function as T1 or T2 MRI contrast agents.

[93] For example, the surface of a superparamagnetic iron oxide nanoparticle may be coordinated to surface-exposed crosslinker-derived amino groups or -COOH functional groups of the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention. In this case, the particle may be used as a T2 contrast agent that makes the corresponding region appear darker in MRI images through magnetic field perturbation within an MRI system.

[94] Accordingly, a drug based on the crosslinked polysaccharide colloidal particles of the present invention may function as an MRI contrast agent, whether the MRI contrast agent itself serves as the drug or a separate drug is conjugated / loaded, wherein iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), manganese ions (Mn'2'), or iron oxide particles are coordinated to surface-exposed crosslinker-derived amino groups or -COOH functional groups of the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention. Thus, following injection into the body, the location of the crosslinked polysaccharide colloidal particle-based drug of the present invention can be tracked by MRI to determine whether it is 21 retained in lymph nodes through interactions with immune cells in lymph nodes where various immune cells gather (FIG. 18); whether it is phagocytosed by macrophages following injection into the body; whether it undergoes metabolic degradation; whether it circulates in the blood; whether it is delivered to cellular parenchyma through capillaries; whether it accumulates in tissues; whether it is excreted in urine through the kidneys; whether it is excreted in feces; whether it is absorbed into the vascular circulatory system following injection into the body; whether it leaks through blood vessel walls; whether it can be collected from urine / feces and reused; and whether it is introduced into cells.

[95] The crosslinked polysaccharide colloidal particle of the present invention having iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) coordinated to surface-exposed crosslinker-derived amino groups or -COOH functional groups may be designed / synthesized such that crosslinker-derived basic amino groups and / or -COOH functional groups to which metal ions are not coordinated remain exposed on its surface. Accordingly, the particle may simultaneously act as a chelator of metal ions (Examples 4 and 5). Thus, a separate chelator need not be used.

[96] The non-immunogenic crosslinked polysaccharide colloidal particle according to the present invention has the following advantages in biocompatibility and diagnostic imaging over existing MRI contrast agents:

[97] (1) Design for immune inactivity: The structure is designed to avoid recognition by PRRs (e.g., TLRs) and / or BCRs on immune cells in the body, thereby suppressing immunogenicity even upon repeat administration.

[98] (2) High dispersion stability and optimal particle size: Uniformly distributed water-soluble crosslinked polysaccharide colloidal particles having a size of about 2-8 nm exhibit prolonged residence time in lesional tissues with minimized immune stimulation and induce an increase in interstitial space (interstitial space enhancement) and / or distension of anatomical spaces (anatomical space distension), thereby enabling uniform contrast enhancement signals to be obtained (FIGS. 11 to 14 and FIG. 20).

[99] (3) Increased lesion specificity in different tissues: Nonspecific accumulation is suppressed in inflammatory lesions, whereas selective accumulation in tumor tissues is possible through increased vascular permeability, such as the EPR effect, thereby improving image contrast resolution.

[100]

[101] [Immune Responses, Immune Cells, and Immunogenicity]

[102] Conventional drug delivery systems have not completely eliminated adverse effects arising from immunogenicity and have had the problem of increased immune-mediated adverse effects upon repeat administration. Accordingly, the present invention seeks to overcome these problems by minimizing binding strength to immune cells and providing a structure resistant to degradation by enzymes in the body.

[103] The non-immunogenic crosslinked polysaccharide colloidal particles of the present invention have a compact, spherical, three-dimensional network structure formed by selectively modifying the -OH functional groups of monosaccharides constituting the repeating structures of various polysaccharide polymers, including dextran, with an epoxide-based first crosslinking agent and chemically crosslinking the polymers. The surface properties resulting from synthesis conditions, such as the type and amount of crosslinking agent, reaction temperature, reaction time, pH, and reactant concentration, are designed to minimize binding strength to PRRs, such as TLRs, and BCRs. Accordingly, provided that the dose is adjusted to prevent gelation or aggregation at the administration site, the particles are not recognized as foreign antigens by surface receptors on immune cells, and immune activation is suppressed even upon repeat administration. In particular, with respect to B cells, the particles are designed to prevent abnormal immune stimulation arising from signal enhancement through BCR clustering, provided that the dose is adjusted to prevent gelation or aggregation at the administration site.

[104] An antigen is a molecule that induces an immune response and may include proteins, DNA, RNA, lipids, polysaccharides, and the like.

[105] In general, when a foreign substance is introduced into the body, it may be recognized by innate immune receptors (PRRs), B-cell receptors (BCRs), or the like, thereby triggering an immune response. Such responses lead to rapid clearance of drug carriers and adverse reactions, as well as reduced efficacy of therapeutic agents or contrast agents.

[106] In particular, in B cells, BCRs cluster within a specific spatial region, determining signal strength and duration. If abnormal immune stimulation consequently occurs, B-cell proliferation, differentiation, and antibody production are promoted, creating a risk of allergy, inflammation, hypersensitivity reactions, and the like.

[107] Meanwhile, polysaccharides may induce immune responses by binding to pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), present on the surfaces of immune cells, particularly macrophages and dendritic cells. This action contributes to enhancing T-cell and B-cell responses to antigens. Specifically, polysaccharides such as dextran may bind to PRRs on immune cells and induce immune cell responses, such as increased phagocytosis by macrophages, increased MHC-II expression and enhanced antigen presentation by dendritic cells, and promoted secretion of cytokines, such as IL-6, TNF-a, and IL-12.

[108] Accordingly, to provide a platform technology based on non-immunogenic crosslinked polysaccharide colloidal particles that are not recognized by immune cells in the body and elicit minimized immune responses, the present invention involves designing and synthesizing non-immunogenic crosslinked polysaccharide colloidal particles as follows. The -OH functional groups of monosaccharides constituting the repeating structures of various polysaccharide polymers, including dextran, which can induce innate immune activation through binding to PRRs and enhance the function of antigen-presenting cells, are selectively modified with an epoxide-based first crosslinking agent, and the polymers are chemically crosslinked to form a compact, spherical, three-dimensional network structure. The surface of this structure is optimized to avoid interactions with PRRs, such as TLRs, or cell receptors, such as BCRs, on immune cells, and to suppress immune activation even upon repeat administration. Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particles according to the present invention, which are not recognized by immune cells in the body and elicit minimized immune responses, may be used in various medical applications, including early diagnosis of inflammatory diseases, lesion-targeted delivery, and development of safe drug delivery systems, such as vaccines.

[109] In the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention, the compact, spherical, three-dimensional network structure formed by (a-1) selectively modifying the -OH functional groups of monosaccharides constituting the repeating structure of the polysaccharide with an epoxide-based first crosslinking agent, (a-2) inducing intramolecular and / or intermolecular crosslinking reactions between functional groups modified with the first crosslinking agent and spatially adjacent -OH functional groups, directly and / or via a second crosslinking agent containing two or more -NH functional groups, and (a-24 3) modifying some or all of the surface-exposed crosslinker-derived basic amino groups into -COOH functional groups to adjust the surface charge within a range of -20 mV to 0 mV,

[110] (i) has markedly reduced binding strength to surface receptors on immune cells in the body, such as PRRs and BCRs, such that the immune response switch of the immune cells is not activated, or the structure cannot induce receptor clustering that initiates a signal transduction cascade by acting as a multivalent antigen having two or more epitopes,

[111] (ii) consequently does not induce B-cell activation or antibody production,

[112] (iii) provides stability against hydrolysis by enzymes in the body and, at the same time,

[113] (iv) can be stably eliminated from the body without hypersensitivity reactions even in pathological environments, such as inflammatory lesions.

[114] The markedly reduced binding strength of the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention to surface receptors on immune cells can be confirmed from MRI images showing that the T1 MRI contrast agent based on the crosslinked polysaccharide colloidal particles of the present invention is transported as far as the central lymphatic vessels without being retained through interactions with immune cells in lymph nodes where various immune cells gather (Example 5-7, FIG. 18).

[115] In addition, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention have markedly reduced binding strength to surface receptors on immune cells, thereby suppressing immune recognition of foreign substances and activation signals, such as the NF-kB pathway (Example 2, FIG. 4). Consequently, B-cell activation and antibody production are not induced even upon repeat administration, markedly reducing immune-mediated adverse effects (minimization of immunogenicity).

[116] In the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, the -OH functional groups of monosaccharides constituting the repeating structure of a polysaccharide polymer are selectively modified with an epoxide-based first crosslinking agent, and the polymer is chemically crosslinked. The crosslinking reaction strengthens the particle structure, thereby inhibiting hydrolysis by enzymes in the body. Owing to optimized particle size and surface charge control, the particles are stably eliminated from the body through normal physiological elimination pathways, such as those involving the kidneys and the lymphatic system (enzyme resistance and stable elimination).

[117] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may be used in various medical applications, including early diagnosis of inflammatory diseases, lesion-targeted delivery, and development of safe drug delivery systems, and, in particular, may be repeatedly administered without adverse effects arising from immune responses.

[118] The composition based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention is a water-soluble polymer platform having a threedimensional network structure designed through selective chemical modification, with a crosslinked structure precisely controlled to minimize interactions with surface receptors, such as PRRs and BCRs, on immune cells in the body. Accordingly, the non-immunogenic drug delivery platform, which can significantly suppress immune system activation even upon repeat administration by selectively modifying the -OH functional groups of monosaccharides with an epoxide-based first crosslinking agent and optimizing crosslinking reaction conditions, such as crosslinking agent type and concentration, reaction time, temperature, and pH, according to the present invention, can minimize inconsistent immune responses arising from interspecies differences in immune receptors even when the same polysaccharide structure is used.

[119] In fact, no immune-related adverse reactions were observed even when the contrast agent based on the crosslinked polysaccharide colloidal particles of the present invention was administered into the body as a drug. In the Phase 1 / 2a clinical trial conducted (Stage 1 Safety Set: n=9; Stage 2 Safety Set: n=23), hematological analyses over 24 hours following a single intravenous injection of NEMO-103, a crosslinked dextran-based T1 contrast agent, showed no significant changes in indicators of systemic inflammatory responses, hypersensitivity reactions, or allergic reactions, such as white blood cell count and C-reactive protein (Example 4-5). This indicates that the contrast agent components can be safely eliminated through physiological excretory pathways without unnecessary interactions with the immune system in the body.

[120] In addition, toxicity studies of INV-001, an iron-based lymphatic contrast agent, in animal models showed no significant toxic responses in clinical signs, such as edema, body weight changes, and organ lesions, hematological parameters, or histopathological findings, despite repeated administration to rats at doses up to 1,500 mg / kg on a dextran basis (Example 5-8, Table 7). This provides important preclinical evidence that the composition based on the 26 non-immunogenic crosslinked polysaccharide colloidal particles of the present invention can maintain biocompatibility and immunological safety even at high doses.

[121] In addition, in a Phase 1 clinical trial, INV-001 consistently enabled high-resolution visualization of lymph nodes, which are densely populated with immune cells. At the same time, the maximum tolerated dose (MTD) was not reached, no adverse drug reactions (ADRs) or serious adverse events (SAEs) occurred, and non-immunogenicity based on low binding strength to immune cell receptors, such as BCRs and PRRs, was confirmed, thereby demonstrating both lymphatic system-specific imaging efficacy and safety based on immune inactivity (Example 6).

[122] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particle composition of the present invention is effective as a non-immunogenic polymer-based drug delivery platform that not only enables precise control of pharmacokinetic behavior in the body but also effectively suppresses adverse immune reactions, such as inflammation, allergy, and hypersensitivity reactions, by minimizing interactions with the immune system. Its potential for various biomedical applications, including T1 MRI contrast agents, is thereby supported.

[123] An immunogen is a substance capable of eliciting an immune response. An antigen refers to any substance, generally a foreign substance, that specifically binds to an antibody or a T-cell receptor. All immunogens are antigens, but antigens such as haptens are not immunogens.

[124] Accordingly, as used herein, “reduced or minimized immunogenicity” may mean that the crosslinked polysaccharide colloidal particle of the present invention does not elicit an immune response as an antigen, does not induce the production of antibodies against itself as an antigen, can be repeatedly administered, does not elicit an inflammatory response, or does not induce a chronic inflammatory disease.

[125] As used herein, a “crosslinked polysaccharide colloidal particle having reduced or minimized immunogenicity toward immune cells relative to the polysaccharide to be crosslinked” may mean that, during distribution in the body, the crosslinked polysaccharide colloidal particle of the present invention is not recognized as a foreign antigen by the immune system; does not induce the production of antibodies against the crosslinked polysaccharide colloidal particle; does not cause hypersensitivity reactions or immune-mediated adverse effects; does not activate the complement system; does not elicit an inflammatory response; is not phagocytosed by macrophages upon repeat administration; is not recognized or bound by 27 pattern recognition receptors (PRRs); does not cause cytokine release; or does not stimulate the NF-kB activation pathway in macrophages.

[126] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be such that it does not activate immune cells, such as B cells and mast cells, or induce their proliferation during distribution in the body.

[127] Generally, when a foreign antigen binds to a cell membrane receptor on an immune cell, either directly or indirectly through an antibody, it may activate the immune cell through signal transduction.

[128] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is designed not to act as an immunogen or antigen that binds directly to surface receptors, such as PRRs and BCRs, on immune cells or indirectly through antibodies to immune cells or non-immune cells bearing Fc receptors.

[129] As used herein, the antibody may be IgM, IgD, IgG, IgA, or IgE.

[130] Binding of a single antibody molecule to an FcR usually does not elicit any response. If a response were elicited by a single antibody alone, the immune system would be excessively activated at all times. Accordingly, a response generally occurs only when multiple immune complexes and Fc receptors are cross-linked.

[131] Immune cells bearing Fc receptors include B lymphocytes, mast cells, basophils, follicular dendritic cells, macrophages, neutrophils, eosinophils, and natural killer (NK) cells. Fc receptors are also present on some non-immune cells, contributing to immune surveillance and inflammatory responses. Non-immune cells bearing Fc receptors include endothelial cells and epithelial cells.

[132] Fc receptors on endothelial cells may mediate transport of immune complexes across cell layers and may play a role in inflammation. Certain epithelial cells express Fc receptors that may participate in immune responses, particularly at mucosal surfaces.

[133] The polysaccharide to be cross-linked in the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be a T-cell-independent multivalent antigen having two or more epitopes. In contrast, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is designed / synthesized so that adverse immune reactions do not occur during distribution in the body, for example, so 28 that it does not induce B-cell activation or antibody production, and specifically so that it does not induce the production of antibodies that recognize or bind to it as a multivalent antigen having two or more epitopes.

[134] Most T-cell-independent antigens, such as polysaccharides, comprise multiple identical epitopes in each molecule. Such multivalent antigens can effectively cross-link numerous B-cell antigen receptors and initiate a response.

[135] Dextran, which is the polysaccharide to be cross-linked in the non-immunogenic crosslinked polysaccharide colloidal particle according to an embodiment of the present invention, is a large polysaccharide capable of displaying multiple epitopes and can therefore bind simultaneously to multiple BCRs on a single B cell. This causes the BCRs to cluster together (cross-linking).

[136] BCR clustering initiates a signal transduction cascade within a B cell. Some activated B cells become memory B cells. These cells remain in the body and may mount a faster and stronger response upon encountering the same antigen again.

[137] Accordingly, after the crosslinked polysaccharide colloidal particle of the present invention is designed / synthesized so that adverse immune reactions do not occur during distribution in the body, specifically so that it does not induce the production of antibodies that recognize or bind to it as a multivalent antigen having two or more epitopes, whether the particle is a multivalent antigen may be determined, for example, by mixing it with antibodies against the polysaccharide to be cross-linked, such as dextran, and assessing whether it binds to the antibodies and, further, whether aggregation occurs through binding between multivalent antigens and antibodies. This may also be determined by assessing whether, following repeat administration of the crosslinked polysaccharide colloidal particles of the present invention, there is an increase in cytokines associated with proliferation upon memory B-cell activation and / or in the number of lymphocytes.

[138] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may reduce or modify / eliminate the combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions that is sufficiently effective to cause adverse immune reactions through interactions with BCRs / antibodies, by selectively modifying the -OH functional groups of monosaccharides constituting the repeating structure of the polysaccharide to be cross-linked with an epoxide-based first crosslinking agent and then 29 forming a compact spherical structure through intramolecular and / or intermolecular crosslinking.

[139] An epitope is a distinct part of an antigen that is recognized by the immune system. In the case of a polysaccharide, an epitope may consist of a specific arrangement or conformation of saccharide residues.

[140] The individual glucose units of a dextran molecule and their specific arrangement are critical for recognition (glucose residues). a-1,6-Glycosidic bonds form the dextran backbone and contribute to its overall shape and epitope display. a-1,3-Branch points may create unique structural motifs recognized by BCRs.

[141] Molecular interaction, namely binding, involves noncovalent interactions, such as hydrogen bonding, van der Waals forces, and hydrophobic interactions, between the CDRs of a BCR and the glucose residues of dextran. The spatial arrangement of glucose units within dextran and the specific conformation of its branches play an important role in binding specificity.

[142] However, when a polysaccharide is chemically crosslinked through intramolecular and / or intermolecular crosslinking by selectively modifying the -OH functional groups of its monosaccharide building blocks with an epoxide-based first crosslinking agent according to the present invention to form compact, nearly spherical nanoparticles, the rotational freedom of chemical bonds is drastically reduced. This makes it difficult to provide a three-dimensional structure capable of specifically binding to a BCR or the antigen-binding site of an antibody through a combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions.

[143] In summary, B-cell receptors (BCRs) and antibodies that recognize dextran do so through their variable regions, particularly their CDRs, which bind to distinct epitopes formed by the glucose residues and specific structural features of dextran. This binding involves a combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions, allowing BCRs / antibodies to specifically recognize and bind to dextran.

[144] Accordingly, the crosslinked polysaccharide colloidal particle according to the present invention is formed by (i) modifying the -OH functional groups of glucose constituting the building blocks of dextran to be cross-linked with a first crosslinking agent having an epoxide group, and forming a crosslinked polysaccharide particle by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2), and (ii) adjusting the number of surface-exposed crosslinker-derived basic amino groups through modification into -COOH functional groups. By forming dextran, a complex branched polysaccharide, into a spherical structure through crosslinking, the particle alters the spatial arrangement of glucose units within dextran and / or the specific conformation of its branches. This may reduce or modify / eliminate the combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions at epitope sites within dextran corresponding to antibodies that bind to dextran.

[145] Specifically, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is formed into a compact spherical structure through chemical intramolecular and / or intermolecular crosslinking after selective modification of the -OH functional groups of monosaccharides constituting the building blocks of the polysaccharide with an epoxide-based first crosslinking agent. This markedly reduces binding strength to surface receptors, such as PRRs and BCRs, on immune cells in the body so that the immune response switch is not activated, and may reduce or minimize the immunogenicity / antigenicity of the polysaccharide to be cross-linked.

[146] As described above, most T-cell-independent antigens, such as polysaccharides, are multivalent antigens comprising multiple identical epitopes in each molecule and can therefore effectively cross-link numerous B-cell antigen receptors and initiate a response even without recognition by helper T lymphocytes. In this case, memory B cells may be formed that remain in the body and can mount a faster and stronger response upon encountering the same antigen again, and repeated injections are therefore not possible because of the development of antibodies.

[147] In contrast, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are designed and synthesized by forming the polysaccharide molecule(s) to be cross-linked into spherical structures through intramolecular and / or intermolecular crosslinking, so as to reduce or modify / eliminate the combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions with BCRs that is sufficiently effective to cause adverse immune reactions, preferably so that the particles do not act as multivalent antigens that initiate B-cell activation (proliferation and differentiation) through antigen-induced clustering of receptors and antigen-mediated cross-linking of the BCR complex. Accordingly, repeat administration may be possible without the development of antibodies against the crosslinked polysaccharide colloidal particles.

[148] For example, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be designed / synthesized to have low avidity for B cells or antibodies upon administration into the body by controlling parameters such as the hydrodynamic diameter of the particle, the molecular weight of the polysaccharide to be cross-linked, the type of polysaccharide, the type or combination of monosaccharides within the polysaccharide, the type of crosslinking agent, modification of one terminus of a surface-exposed crosslinking agent, and crosslinking density.

[149] The crosslinked polysaccharide colloidal particles of the present invention do not act as T-cell-independent multivalent antigens that initiate B-cell activation (proliferation and differentiation) through antigen-induced clustering of receptors and antigen-mediated crosslinking of the BCR complex, and blood tests performed 24 hours after administration in the safety evaluation of the Phase 1 / 2a clinical trial (Example 4-5) showed no increase in the number of lymphocytes, such as B cells. In addition, analyses of hematological tests in a 4-week repeated intradermal administration toxicity study of INV-001 in beagle dogs (once every 2 weeks, for a total of 3 administrations; Example 5-8, Table 7) and hematological tests during the Phase 1 / 2a clinical trial of shoulder MR arthrography using NEMO-103, a crosslinked dextran-based T1 MRI contrast agent (Example 4-5), confirmed that, following administration of the crosslinked polysaccharide colloidal particles themselves into the body, there were no clinically significant abnormal changes in total blood cell count, neutrophil count, eosinophil count, basophil count, or monocyte count. Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may be designed / synthesized to permit repeat administration.

[150] The non-immunogenic crosslinked polysaccharide colloidal particle according to the present invention is characterized in that, upon administration into the body, it does not elicit one or more adverse immune reactions selected from the group consisting of inflammation, edema, allergy, and hypersensitivity reactions.

[151] Clinical manifestations of hypersensitivity reactions include urticaria, a localized skin reaction accompanied by itching and edema; angioedema, swelling of the deeper layers of the skin around the eyes and lips; and anaphylaxis, a severe systemic allergic reaction that may include symptoms such as difficulty breathing, hypotension, and shock.

[152] FIG. 2 shows that, unlike Dextran T10, the polysaccharide to be cross-linked, NEMO-103 prepared using crosslinked dextran (Cdex) did not cause an acute edema response in vivo.

[153] Analyses of hematological tests in a 4-week repeated intradermal administration toxicity study of INV-001 in beagle dogs (once every 2 weeks, for a total of 3 administrations; Example 5-8, Table 7) and hematological tests during the Phase 1 / 2a clinical trial of shoulder MR arthrography using NEMO-103, a crosslinked dextran-based T1 MRI contrast agent (Example 4-5), confirmed that, following administration of the crosslinked polysaccharide colloidal particles themselves into the body, there were no clinically significant abnormal changes in total blood cell count, neutrophil count, eosinophil count, basophil count, or monocyte count. It can therefore be inferred that the crosslinked polysaccharide colloidal particles of the present invention themselves do not cause inflammatory responses or hypersensitivity reactions in the body.

[154] In addition, according to Example 3-12 and FIG. 10, imaging of the route and distribution of the T1 MRI contrast agent based on the crosslinked polysaccharide colloidal particles according to the present invention following administration into the body shows, for example, that the agent retains its performance as a T1 MRI contrast agent during circulation through the vascular system until excretion from the body in urine. This observation, in combination with analyses of hematological tests in a 4-week repeated intradermal administration toxicity study of INV-001 in beagle dogs (once every 2 weeks, for a total of 3 administrations; Example 5-8, Table 7) and hematological tests during the Phase 1 / 2a clinical trial of shoulder MR arthrography using NEMO-103, a crosslinked dextran-based T1 MRI contrast agent (Example 4-5), can confirm that the agent was not recognized as a foreign substance and that the body did not elicit an inflammatory response to eliminate it.

[155] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be such that it is not recognized or bound by pattern recognition receptors (PRRs), does not cause cytokine release, or does not stimulate the NF-kB activation pathway in macrophages (FIGS. 4 and 6).

[156] Representative PRRs include Toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (retinoic acid-inducible gene-I-like receptors, RLRs), NOD-like receptors (nucleotide oligomerization domain-like receptors, NLRs), and AIM-like receptors (absent-inmelanoma-like receptors, ALRs).

[157] As shown in FIG. 4, Cdex itself did not stimulate the NF-kB activation pathway when applied to macrophages. As shown in FIG. 6, Cdex itself did not affect cell viability even when applied at a high concentration. These results suggest that Cdex itself does not induce intracellular signaling-mediated activation of macrophages, such as cell proliferation and cytokine release.

[158] Accordingly, analyses of hematological tests in a 4-week repeated intradermal administration toxicity study of INV-001 in beagle dogs (once every 2 weeks, for a total of 3 administrations; Example 5-8, Table 7) and hematological tests during the Phase 1 / 2a clinical trial of shoulder MR arthrography using NEMO-103, a crosslinked dextran-based T1 MRI contrast agent (Example 4-5), confirmed that, following administration of the non-immunogenic crosslinked polysaccharide colloidal particles themselves into the body, there were no clinically significant abnormal changes in total blood cell count, neutrophil count, lymphocyte count, or monocyte count. From this, it can be inferred that the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are not recognized or bound by PRRs, or do not cause cytokine release.

[159] The linear polysaccharide, branched polysaccharide, or cyclic polysaccharide to be crosslinked may be a homopolysaccharide or a heteropolysaccharide.

[160] Non-limiting examples of linear polysaccharides, branched polysaccharides, or cyclic polysaccharides include dextran, cyclodextrin, maltodextrin, and inulin, as illustrated in FIG. 8.

[161] As used herein, dextran also includes various derivatives thereof. Non-limiting examples of dextran derivatives include carboxymethyl dextran (CM dextran), dextran sulfate, and diethylaminoethyl dextran (DEAE-dextran).

[162] For renal excretion, the dextran or dextran derivative used may have an average molecular weight of 10,000 Da or less, and the spherical crosslinked dextran-based nanoparticles formed by crosslinking dextran-based molecules with a crosslinking agent may have a molecular weight of 90,000 Da or less.

[163] The non-immunogenic crosslinked polysaccharide colloidal particle according to an embodiment of the present invention is a nanoparticle formed by intramolecular and / or intermolecular crosslinking of a complex branched polysaccharide with a crosslinking agent at the -OH functional groups of its monosaccharide building blocks in an aqueous solution. Dimers to trimers may be formed through intermolecular crosslinking of the branched polysaccharide, or a crosslinked polysaccharide colloidal particle may be formed by controlling crosslinking so that only one branched polysaccharide molecule undergoes intramolecular crosslinking with a crosslinking agent. The degree of compression may be controlled by adjusting the degree of crosslinking and / or the molecular weight of the branched polysaccharide, and the particle may preferably have a compact spherical shape.

[164] In particular, an aqueous polymer solution becomes viscous as its concentration increases. The same applies to an aqueous solution containing crosslinked polysaccharide colloidal particles.

[165] For example, when dextran-based molecules having an average molecular weight of 10,000 Da or less are crosslinked with a crosslinking agent at the -OH functional groups of their glucose building blocks in an aqueous solution, one to three dextran-based molecules form spherical nanoparticles through intramolecular and intermolecular crosslinking. Accordingly, the crosslinked dextran-based nanoparticles of the present invention prepared using dextran-based molecules having an average molecular weight of 10,000 Da or less have a molecular weight of 90,000 Da or less, and a complex for injection into the body modified with these nanoparticles may be provided as a nanostructure having a hydrodynamic diameter of 10 nm or less, preferably 5 nm or less, for renal excretion. Generally, a nanostructure must have a hydrodynamic diameter of 6-8 nm or less to permit natural elimination through the kidneys.

[166] Accordingly, by variously designing the synthesis of non-immunogenic crosslinked polysaccharide colloidal particles, including crosslinked dextran-based nanoparticles, and methods of modification using these particles, the biodistribution and elimination of functional nanoparticles or drugs to be modified can be controlled without immune rejection reactions or toxicity. In some cases, even when the modified functional nanoparticles or drugs are eliminated from the body together with the bound non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, they remain stable in plasma without aggregation and are not metabolized or degraded in the body following injection, and may therefore be collected from urine or feces and reused. In some cases, a drug may be separated and released from the non-immunogenic crosslinked polysaccharide colloidal particle in the body, and the drug and particle may each be released within the body through different routes. In this case, the separated non-immunogenic crosslinked polysaccharide colloidal particle is not metabolized or degraded in the body and may therefore be collected from urine or feces and reused. For example, the crosslinked polysaccharide colloidal particle separated from the non-immunogenic crosslinked polysaccharide colloidal particle-based drug of the present invention following injection into the body may be designed to be absorbed into the blood circulatory system and excreted in urine through the kidneys without leakage through blood vessel walls (extravasation).

[167]

[168] [Non-immunogenic Crosslinked Polysaccharide Colloidal Particles in Which 60% or More of the Total Number of Monosaccharide Building Blocks Are Modified with a Crosslinking Agent]

[169] The three-dimensional network formed upon chemical crosslinking of a polysaccharide exhibits subtle differences in the degree of crosslinking, molecular weight distribution, and arrangement of functional groups depending on synthesis conditions, such as the type and amount of crosslinking agent, reaction temperature, reaction time, pH, and reactant concentration. These differences may cause changes in solubility, stability, and interactions in vivo. In addition, particle size, surface charge, porosity, and the like directly affect the biodistribution, residence time in the body, and immunogenicity of drugs based on crosslinked polysaccharide colloidal particles. Differences in binding to cell receptors and tissue distribution depending on the degree of crosslinking must be considered.

[170] The innate immune system comprises cells that recognize pathogens nonspecifically and respond rapidly, including macrophages, dendritic cells, neutrophils, and natural killer (NK) cells. These cells express different pattern recognition receptors (PRRs) and provide an initial defense against foreign pathogens. These receptors recognize saccharide structures present on pathogen surfaces and may induce cell activation and inflammatory responses.

[171] The immune system recognizes repetitive polysaccharide structures and induces immune responses. Accordingly, a drug based on non-immunogenic polysaccharide colloidal particles that are highly chemically crosslinked by selectively modifying the -OH functional groups of monosaccharides constituting the repeating structure of the polysaccharide with an epoxide- based first crosslinking agent according to the present invention may block recognition by pattern recognition receptors (PRRs) by forming a highly dense three-dimensional network and thereby controlling structural repetitiveness and functional group exposure.

[172] Representative PRRs include TLR4 (Toll-like receptor 4), which recognizes microbial polysaccharides such as lipopolysaccharide (LPS); the mannose receptor (MR), which recognizes pathogen-derived mannose structures; and C-type lectin receptors (CLRs), which are cell membrane receptors that recognize polysaccharide structures and play a central role in responses to fungi and some viruses. The present invention prevents activation mediated by immune cells by controlling the degree of crosslinking to limit exposure of repetitive saccharide structures recognizable by these PRRs.

[173] B-cell activation requires sufficient cross-linking of B-cell receptors (BCRs). However, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention have structural constraints that prevent them from inducing cross-linking mediated by BCRs, owing to their highly chemically crosslinked structure formed by selectively modifying the -OH functional groups of monosaccharides constituting the repeating structure of the polysaccharide with an epoxide-based first crosslinking agent. This does not induce B-cell clustering or memory B-cell formation even upon repeat administration, resulting in a markedly low risk of long-term immune responses or antibody production. In other words, the crosslinked structure of the present invention is designed so that it is not recognized as a multivalent antigen and has non-immunostimulatory properties.

[174] According to the present invention, selective modification of the -OH functional groups of a polysaccharide with an epoxide-based first crosslinking agent and crosslinking at high density restrict the spatial arrangement (accessibility) of repetitive saccharide structures and suppress exposure of immune recognition epitopes on the polysaccharide surface. This markedly reduces binding affinity for surface receptors on immune cells, such as PRRs, including TLRs, C-type lectin receptors, and mannose receptors, and BCRs, thereby effectively blocking immune recognition by surface receptors on immune cells. Consequently, the non-immunogenic particles of the present invention are not recognized as multivalent antigens having two or more epitopes and do not induce B-cell receptor clustering or memory B-cell formation, such that immune responses are not induced even upon repeat administration.

[175] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is characterized in that 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent. As a result, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention can not only form a compact spherical structure but also efficiently reduce or minimize immunogenicity toward immune cells relative to the polysaccharide to be cross-linked. In addition, because it is not hydrolyzed by enzymes in the body, immunogenicity attributable to sites exposed through hydrolysis can also be minimized.

[176] Here, in the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention, in which the -OH groups of a polysaccharide dispersed in an aqueous solvent are modified with a crosslinking agent, preferably at least one -OH group in at least one of two consecutive monosaccharides within the polysaccharide is predominantly modified with a crosslinking agent, whereby the particle is not hydrolyzed by enzymes in the body and can have reduced or minimized immunogenicity toward immune cells.

[177] In this regard, the T1 MRI contrast agent according to the present invention is based on non-immunogenic crosslinked polysaccharide colloidal particles formed in an aqueous solvent by (i) modifying the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, followed by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2). Imaging of its route and distribution following administration into the body shows, for example, that the agent retains its performance as a T1 MRI contrast agent during circulation through the vascular system until excretion from the body in urine (Example 3-12 and FIG. 10), from which it can be inferred that the agent has not been hydrolyzed by enzymes in the body. Accordingly, it can be inferred that the agent can be eliminated from the body without exposure of potentially immunogenic saccharide-based sites through hydrolysis and that the resulting immunogenicity is also minimized.

[178] Specifically, in the animal study in Example 3 (intravenous administration), the Phase 1 / 2a clinical trial in Example 4-5 (musculoskeletal contrast agent), and the animal study in 38 Example 5-8 (lymphatic contrast agent), no inflammatory response occurred during distribution in the body of the T1 MRI contrast agent based on the crosslinked polysaccharide colloidal particles of the present invention. MRI images confirmed that the agent was excreted in urine through the kidneys as a result of not being phagocytosed by macrophages or hydrolyzed by enzymes in the body.

[179] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is a water-soluble, amorphous colloidal nanoparticle having a more complex threedimensional network formed by intramolecular and / or intermolecular crosslinking, with a crosslinking agent, of not only a linear polysaccharide, such as inulin, dispersed in an aqueous solvent, but also a complex branched polysaccharide, such as dextran, or a cyclic polysaccharide, such as cyclodextrin, dispersed in an aqueous solvent. Nevertheless, as described above, by modifying with a crosslinking agent 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide dispersed in an aqueous solvent, or by predominantly modifying with a crosslinking agent at least one -OH functional group in at least one of two consecutive monosaccharides within the polysaccharide chain, the particle (i) is not hydrolyzed by enzymes in the body and (ii) has markedly reduced binding strength to surface receptors, such as PRRs and BCRs, on immune cells in the body, such that the immune response switch is not activated. Consequently, immunogenicity toward immune cells can be minimized so that B-cell activation and antibody production are not induced.

[180] Specifically, in the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention, 50% or more of the monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent, thereby markedly reducing binding strength to surface receptors, such as PRRs and BCRs, on immune cells in the body and ensuring that unwanted immune responses are not elicited even when a certain degree of interaction with body tissues or fluids occurs.

[181] Preferably, by more precisely controlling the crosslinking density at a high level according to the molecular weight of the polysaccharide, the particle may not only resist hydrolysis by enzymes in the body but also remain unhydrolyzed at the acidic pH of the stomach or intracellular lysosomes.

[182] The polysaccharide to be cross-linked may be biodegradable through hydrolysis by enzymes in the body. However, the crosslinked polysaccharide colloidal particle of the present invention, formed in an aqueous solvent by (i) modifying the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, and forming a crosslinked polysaccharide particle by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2), and (ii) adjusting the number of surface-exposed crosslinker-derived basic amino groups through modification into -COOH functional groups, may remain unhydrolyzed by enzymes in the body.

[183] Non-limiting examples of enzymes in the body include glycoside hydrolases, which hydrolyze glycosidic bonds in polysaccharides to break them down into simple sugars.

[184] The crosslinked polysaccharide colloidal particle having minimized immunogenicity toward immune cells according to the present invention may have markedly reduced binding strength to surface receptors, such as PRRs and BCRs, on immune cells in the body, such that it is not recognized by immune cells, does not cause cytokine release, or does not stimulate the NF-kB activation pathway, but is not limited thereto.

[185] It can be seen that, even when the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are formed by crosslinking complex branched polysaccharides having high immunogenicity, in addition to linear or cyclic polysaccharides, the particles are not recognized or bound by pattern recognition receptors (PRRs) on immune cell surfaces when 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent (Examples 4-2 and 5-7).

[186] The degree of immunogenicity of the crosslinked polysaccharide colloidal particle of the present invention may vary depending on the degree of crosslinking, the size of the polysaccharide molecule, and the particular chemical modification.

[187] Accordingly, by controlling the degree of crosslinking and the structural / chemical properties of the polysaccharide, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention can have markedly reduced binding strength to PRRs on the surfaces of immune cells in the body, thereby minimizing immunogenicity in applications such as drug delivery systems.

[188] In summary, a drug based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention can minimize immunogenicity toward immune cells by avoiding immune recognition through markedly reduced binding strength to surface receptors, such as PRRs and BCRs, on immune cells in the body.

[189] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be designed to minimize adverse immune reactions in the human body, for example, so as not to elicit immediate and nonspecific immune activation in the human body, by modifying with a crosslinking agent 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide, such that -OH groups in most monosaccharides are modified with the crosslinking agent, while more precisely controlling the intramolecular crosslinking density and / or intermolecular crosslinking density, thereby markedly reducing binding strength to surface receptors, such as PRRs and BCRs, on immune cells in the body and minimizing immunogenicity toward immune cells.

[190] Here, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be formed by intermolecular crosslinking, in an aqueous solvent, of one to three molecules in the case of a linear or branched polysaccharide, or one to thirty molecules in the case of a cyclic polysaccharide.

[191]

[192] [Crosslinked  Polysaccharide Colloidal Particles Designed as Non immunostimulatory Particles—Drug Carriers]

[193] With the aim of minimizing immune responses and associated adverse effects, such as inflammation and hypersensitivity reactions, that may be elicited by foreign substances administered into the body, the present invention provides water-soluble crosslinked polysaccharide colloidal particles having non-immunostimulatory properties and biocompatibility.

[194] The particles of the present invention are formed by selectively modifying the -OH functional groups of a polysaccharide with an epoxide-based first crosslinking agent, followed by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2). Subsequently, some or all of the surface-exposed amino groups (-NH2) are converted into -COOH functional groups to adjust the surface charge of the particles to a range of -20 mV to 0 mV, thereby simultaneously providing evasion of detection by immune cells and resistance to enzymatic degradation. Through this design, the colloidal particles of the present invention are not readily hydrolyzed by enzymes in the body and may be stably eliminated from the body without immune cell activation even in pathological inflammatory environments.

[195] The non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are designed to minimize the likelihood of recognition by pattern recognition receptors (PRRs) expressed by innate immune cells, such as macrophages, dendritic cells, and neutrophils. Consequently, the particles selectively reach inflammatory lesions without stimulating immune cells and are effectively eliminated without retention in tissues, thereby increasing their potential use as imaging contrast agents or therapeutic adjuncts (Example 7).

[196] The colloidal particles of the present invention may extend the therapeutic window by prolonging circulation time (circulation half-life) through evasion of the immune system. Immune-mediated adverse effects, such as inflammation and hypersensitivity reactions, are suppressed. Their ability to evade phagocytosis by immune cells enables targeted drug delivery to specific tissues or cells.

[197] In the non-immunogenic crosslinked polysaccharide particles of the present invention, 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides are modified with a crosslinking agent to form a highly stabilized threedimensional network structure. According to Example 3, upon intravenous injection, these particles were confirmed to be filtered in the kidneys via the blood vessels of the nephrons and excreted in urine, as confirmed by MRI of the bladder, indicating safe elimination from the body without accumulation.

[198] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention have potential for various biomedical applications as drug carriers, imaging contrast agents, therapeutic adjuncts that evade the immune system, and the like. In particular, they are useful as delivery platforms for inflammatory diseases, autoimmune diseases, or vaccines.

[199]

[200] [Non-immunogenic Crosslinked Polysaccharide Colloidal Particles Formed by Crosslinking Polysaccharides so as Not to Pass Through the Walls of Normal Capillaries]

[201] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is formed in an aqueous solvent by (i) modifying the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, and forming a crosslinked polysaccharide particle by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2), and (ii) adjusting the number of surface-exposed crosslinker-derived basic amino groups through modification into -COOH functional groups. The particle may be designed to have a hydrodynamic diameter of 2 to 20 nm, in which case it does not pass through the walls of normal capillaries. In addition, when the hydrodynamic diameter is 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, the particle may (a) be filtered in the kidneys but not pass through the walls of normal capillaries and optionally (b) be drained exclusively into peripheral lymphatic vessels without penetrating or being drained into capillaries at the injection site.

[202] Because the hydrodynamic diameter of the crosslinked polysaccharide colloidal particle increases as the molecular weight of the linear polysaccharide, branched polysaccharide, or cyclic polysaccharide used to synthesize the particle increases, the hydrodynamic diameter of the crosslinked polysaccharide colloidal particle of the present invention can also be controlled within a range of 2 to 20 nm.

[203] The crosslinked polysaccharide colloidal particle of the present invention, designed to have a hydrodynamic diameter of 2 to 20 nm so as not to pass through the walls of normal capillaries, may be formed by crosslinking one to three linear or branched polysaccharide molecules or one to thirty cyclic polysaccharide molecules using a first crosslinking agent having an epoxide group and a second crosslinking agent having two or more amino groups ( NH2).

[204] To allow the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention to (a) be filtered in the kidneys but not pass through the walls of normal capillaries and optionally (b) be drained exclusively into peripheral lymphatic vessels without penetrating or being drained into capillaries at the injection site, its hydrodynamic diameter must be adjusted to 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less.

[205] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may provide a drug modality, such as an imaging modality of a contrast agent, that is drained into peripheral lymphatic vessels when injected into a tissue site rather than administered intravenously.

[206] When the non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is designed to have a hydrodynamic diameter of 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, so as to be filtered in the kidneys, it can pass through the walls of capillaries in the liver and spleen. Accordingly, in this case, capillaries in the liver and spleen are excluded from the normal capillaries through which the crosslinked polysaccharide colloidal particle of the present invention cannot pass.

[207] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is designed to have a hydrodynamic diameter of 2 nm or more and surface-exposed -COOH groups providing a surface charge of -20 mV to 0 mV, so as not to pass through the walls of normal capillaries.

[208] Normal capillaries generally have a diameter of 5-10 pm and thin walls comprising a single layer of endothelial cells, with a functional structure that permits selective exchange of substances, including oxygen, carbon dioxide, nutrients, and metabolites. Tight junctions between the endothelial cells precisely regulate permeability and play an essential role in maintaining homeostasis between blood and tissues.

[209] The non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are characterized by an inability to pass through normal capillary endothelial walls owing to their average particle size and physicochemical properties. Accordingly, tissue penetration is possible only under pathological conditions involving abnormal structural changes in capillaries. These characteristics provide a useful platform for early diagnosis and targeted treatment of diseases associated with pathological changes in capillaries, such as loosening or damage of junctional structures between endothelial cells, inflammatory responses, and hypoxia-induced angiogenesis.

[210] Specifically, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention do not pass through normal capillaries following intravenous injection and exhibit selective infiltration under pathological conditions. Accordingly, the particles of the present invention may be usefully employed as intravenously administered imaging contrast agents or drug carriers and may be applied to diseases in which increased capillary permeability is observed, such as capillary leak syndrome, diabetic microangiopathy (diabetic microvascular complications), thrombotic disease (thrombosis), and pathological angiogenesis in chronic hypoxia or tumor environments.

[211] In particular, the intratumoral environment is characterized by tissue hypoxia and abnormal vascular permeability, and the colloidal particles of the present invention may be applied to various uses, such as anticancer agents, immunomodulators, or MRI contrast agents, based on their physicochemical properties capable of promoting selective accumulation in tumors.

[212] The non-immunogenic crosslinked polysaccharide colloidal particles according to the present invention are designed to selectively infiltrate only lesional tissues without infiltrating normal tissues and are useful as a biomedical platform technology capable of detecting pathological vascular changes in vivo and improving diagnostic and therapeutic efficiency on that basis.

[213] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is designed to have a hydrodynamic diameter of about 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, and a surface charge adjusted to a range of -20 mV to 0 mV. Consequently, the particle is filtered through renal glomerular capillaries but does not pass through the walls of normal capillaries having a normal continuous endothelial structure (continuous endothelium with tight junctions and basal lamina). As a result, the particles of the present invention are selectively drained exclusively into lymphatic vessels and are rapidly excreted in urine following tissue infiltration, thereby minimizing long-term accumulation in the body and associated toxicity (see Examples 3 and 4).

[214] For example, the particles of the present invention are excreted in urine through the renal filtration mechanism following systemic circulation in the bloodstream, and the time required for excretion may therefore be 48 hours or less, preferably 24 hours or less. The non-immunogenic water-soluble crosslinked polysaccharide colloidal particles of the present invention are neither metabolized nor accumulated in the liver and therefore do not cause hepatotoxicity, and also exhibit resistance to phagocytosis by macrophages and hydrolysis by enzymes in the body (see FIGS. 9 and 10).

[215] Using various linear, branched, or cyclic polysaccharides as starting materials, the -OH functional groups of monosaccharides are selectively modified with a first crosslinking agent having an epoxide group, after which particles are formed through intramolecular and / or intermolecular crosslinking reactions between adjacent -OH groups or via a second crosslinking agent comprising amino groups (-NH2). Subsequently, residual surface-exposed amino groups are modified into -COOH functional groups to impart a negative charge, thereby precisely controlling the surface charge between -20 mV and 0 mV (see Example 1-16 and Example 3).

[216] Examples 5, 6, and 7 confirmed that the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention do not pass through healthy vascular endothelium and are selectively transported or drained only under pathological conditions or within the lymphatic circulatory system, in particular being effectively drained through peripheral lymphatic vessels. In addition, no retention of INV-001 at the injection site or within lymph nodes or lymphatic vessels was observed at 24 and 48 hours following intravenous administration. MRI images (FIG. 18) confirmed that INV-001 was transported as far as the central lymphatic vessels without being retained through interactions with immune cells in lymph nodes where various immune cells gather (Example 5-7). This demonstrates rapid excretion and suppression of accumulation in the body.

[217] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention offer potential use as drugs for diagnosis and monitoring of lymphatic diseases and for contrast enhancement in imaging, based on four biological properties: (i) tissue-selective blockage of permeation and lymphatic drainage, (ii) renal excretion, (iii) evasion of hepatic metabolism, and (iv) immune evasion in the body and stability against enzymes (FIG. 20).

[219] [Method for Preparing Non-immunogenic Crosslinked Polysaccharide Colloidal Particles]

[220] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be a crosslinked polysaccharide colloidal particle formed by intramolecular and / or intermolecular crosslinking, with a crosslinking agent at the -OH functional groups of the monosaccharide building blocks, of one to three linear or branched polysaccharide molecules or two to thirty cyclic polysaccharide molecules to be cross-linked dispersed in an aqueous solvent.

[221] As a non-limiting example, the crosslinked polysaccharide colloidal particle of the present invention may be provided by a preparation method comprising the following steps:

[222] a first step of preparing an aqueous solution of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide;

[223] a second step of adding dropwise an alkaline aqueous solution and a first crosslinking agent having an epoxide functional group that reacts with hydroxyl groups (-OH) of the polysaccharide, thereby modifying the -OH functional groups of monosaccharides constituting the building blocks of the polysaccharide with the first crosslinking agent;

[224] a third step of adding dropwise a second crosslinking agent having two or more amino groups (-NH2), thereby causing spatially adjacent functional groups modified with the first crosslinking agent to undergo intramolecular and / or intermolecular crosslinking via the second crosslinking agent and producing crosslinked polysaccharide colloidal particles having terminal amino groups derived from the second crosslinking agent on their surfaces;

[225] a fourth step of adding an organic acid anhydride to the crosslinked polysaccharide colloidal particles having terminal amino groups derived from the second crosslinking agent on their surfaces, thereby modifying some or all of the terminal amino groups into carboxylic acid groups and / or carboxylate groups; and

[226] optionally, a fifth step of adding a precursor, such as iron chloride, that provides iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+), or an aqueous solution of iron oxide nanoparticles, to the water-dispersible crosslinked polysaccharide colloidal particles prepared in the preceding step, thereby preparing (i) crosslinked polysaccharide colloidal particles having iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) coordinated to surface-exposed crosslinker-derived amino groups or -COOH functional groups, or (ii) a complex in which the surfaces of iron oxide nanoparticles are modified with crosslinked polysaccharide colloidal particles.

[227] In the present invention, linear polysaccharides, branched polysaccharides, or cyclic polysaccharides are subjected to intramolecular and / or intermolecular crosslinking in an aqueous solution to prevent unintended increases in hydrodynamic size caused by swelling or the like of the polysaccharide molecules, prevent hydrolysis by enzymes in the body, and reduce or minimize immunogenicity toward immune cells relative to the polysaccharide to be cross-linked, thereby allowing elimination from the body without exposure of potentially immunogenic saccharide-based sites.

[228] It was found that, when a branched polysaccharide such as dextran is crosslinked with a crosslinking agent, two to three branched polysaccharide molecules form a spherical core through intramolecular and intermolecular crosslinking. Similarly, intramolecular and intermolecular crosslinking of two to three molecules in the case of a linear polysaccharide, or up to thirty molecules in the case of a cyclic polysaccharide, enabled the provision of non-immunogenic crosslinked polysaccharide colloidal particles having a hydrodynamic size that prevents passage through the walls of normal capillaries while permitting filtration through renal capillaries.

[229] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is a spherical, amorphous crosslinked polysaccharide colloidal particle formed by modifying the -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, followed by intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2).

[230] For the non-immunogenic crosslinked polysaccharide colloidal particles, the number of linear, branched, or cyclic polysaccharide molecules to be cross-linked may be controlled to be the same or different through synthesis conditions and / or purification.

[231] For example, in the present invention, through the second and third steps, (i) a first crosslinking agent having an epoxide group that reacts with hydroxyl groups (-OH) of the polysaccharide and a functional group that chemically bonds to hydroxyl groups (-OH) and / or amino groups (-NH2), and (ii) a second crosslinking agent having two or more amino groups (-NH2) are added to an aqueous solution of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide and allowed to react. This modifies and crosslinks the polysaccharide with the crosslinking agents at the -OH functional groups of its monosaccharide building blocks, thereby enabling preparation of non-immunogenic crosslinked polysaccharide colloidal particles in which 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent.

[232] In the second step, the first crosslinking agent, which has an epoxide group that reacts with hydroxyl groups (-OH) and a functional group that chemically bonds to hydroxyl groups (-OH) and / or amino groups (-NH2), is not limited in type, provided that it modifies the -OH functional group sites of monosaccharides to permit a reaction (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent and the second crosslinking agent. Preferably, the first crosslinking agent may be a haloalkyl oxirane, for example, epichlorohydrin.

[233] In the third step, the second crosslinking agent having two or more amino groups (-NH2) may be replaced with any crosslinking agent capable of forming a covalent bond with a functional group derived from the first crosslinking agent that has modified an -OH functional group site of a monosaccharide, and such replacement also falls within the scope of the present invention.

[234] In an embodiment of the present invention, when epichlorohydrin is used as the first crosslinking agent for modifying the -OH functional groups of monosaccharides, ethylenediamine or diethylenetriamine (DETA) may be used as the second crosslinking agent that participates in crosslinking between spatially adjacent modified functional groups.

[235] As illustrated in FIGS. 1a and 1b, when a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide reacts with epichlorohydrin as the first crosslinking agent and ethylenediamine or diethylenetriamine (DETA) as the second crosslinking agent, a series of chemical modifications occurs that substantially changes the structure and properties of the polysaccharide.

[236] According to the present invention, modification of a polysaccharide using epichlorohydrin as the first crosslinking agent in combination with ethylenediamine or diethylenetriamine (DETA) as the second crosslinking agent increases crosslinking and improves stability.

[237] In particular, terminal amino groups derived from the second crosslinking agent and exposed on the surfaces of the crosslinked polysaccharide colloidal particles may impart new properties, such as enhanced chelating ability, increased reactivity, and the potential for further functionalization. Accordingly, the crosslinked polysaccharide colloidal particles produced in the third step, which have terminal amino groups derived from the second crosslinking agent on their surfaces, may have an enhanced ability to interact with other molecules, such as through chelation or drug conjugation, via the amino groups (-NH2).

[238] The crosslinked polysaccharide colloidal particles formed in the third step and having surface-exposed terminal amino groups derived from the second crosslinking agent may be modified in the fourth step to expose -COOH functional groups on their surfaces, thereby adjusting the surface charge to -20 mV to 0 mV and allowing a biocompatible pH to be achieved. In addition, the number of crosslinker-derived amino groups exposed on the surfaces of the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may be controlled through a reaction that introduces -COOH-containing functional groups.

[239] For example, when surface-exposed crosslinker-derived amino groups of the crosslinked polysaccharide colloidal particles are modified into -COOH-containing functional groups, the surface charge can be freely controlled within a range of -20 mV to 0 mV by adjusting the type of -COOH-containing functional group and / or the degree of modification. In the fourth step, for example, the surface charge can be freely controlled within a range of -20 mV to 0 mV by adjusting the amount of succinyl anhydride (SA). As the amount of added SA increases, the surface charge becomes more negative.

[240] When surface-exposed crosslinker-derived amino groups of the crosslinked polysaccharide colloidal particles of the present invention are replaced with carboxyl groups, their interactions with water and other molecules change.

[241] For example, in the fourth step, the number of crosslinker-derived amino groups exposed on the surfaces of the crosslinked polysaccharide particles of the present invention may be controlled by adjusting the amount of organic acid anhydride added. Accordingly, the number of functional molecules, such as drugs or targeting molecules, bound to the crosslinked polysaccharide colloidal particles may also be controlled by adjusting the reaction ratio with the drug.

[242] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention may be bound to functional nanoparticles, molecules, or metal ions through covalent or coordination bonds via surface-exposed crosslinker-derived amino groups and / or -COOH functional groups.

[243] The non-immunogenic crosslinked polysaccharide colloidal particle of the present invention is capable of forming strong coordination bonds between crosslinker-derived functional groups, namely carboxyl groups or amino groups, and iron oxide nanoparticles, thereby imparting colloidal stability.

[244] In the fifth step, non-limiting examples of hydrophilic functional groups that coordinate with iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), manganese ions (Mn2+), or iron oxide nanoparticles include hydroxyl groups, carboxylic acid groups, carboxylate groups, and amino groups.

[245] The non-immunogenic crosslinked polysaccharide colloidal particles coordinated with iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) according to the present invention may be designed and synthesized through the first to fifth steps to function as T1 MRI contrast agents that produce bright signals in MRI images. Accordingly, the locations of the crosslinked polysaccharide colloidal particles may be tracked by MRI following injection into the body.

[246] The non-immunogenic crosslinked polysaccharide colloidal particles according to the present invention may be designed and synthesized to maintain their contrast enhancement effect for a relatively long time, thereby allowing longer MRI scan times to improve spatial resolution and enabling imaging at higher resolution. Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particles according to the present invention enable MRI visualization of lymphatic vessels in vivo without venous contamination, although these vessels are clinically highly important and difficult to visualize using existing contrast agents.

[247] In summary, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may also function as T1 or T2 MRI contrast agents when iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) are coordinated to their surface-exposed crosslinker-derived functional groups.

[248] In addition, metal oxide nanoparticles, such as iron oxide nanoparticles, designed to function as T1 or T2 MRI contrast agents may have their surfaces modified with non-immunogenic crosslinked polysaccharide colloidal particles in the fifth step. This allows them to move between structures in the body without aggregation upon injection into the body and thus perform their intended function as T1 or T2 MRI contrast agents.

[249] Accordingly, because the non-immunogenic crosslinked polysaccharide colloidal particles according to the present invention may function as MRI contrast agents, the location of a drug based on the non-immunogenic crosslinked polysaccharide colloidal particles may be tracked by MRI following injection into the body to determine whether it is selectively drained into peripheral lymphatic vessels without venous contamination; whether it is phagocytosed by macrophages following injection into the body; whether it undergoes metabolic degradation; whether it circulates in the blood; whether it is delivered to cellular parenchyma through capillaries; whether it accumulates in tissues; whether it is excreted in urine through the kidneys; whether it is excreted in feces; whether it is absorbed into the vascular circulatory system following injection into the body; whether it leaks through blood vessel walls; whether it can be collected from urine / feces and reused; and whether it is introduced into cells.

[250] Meanwhile, the non-immunogenic crosslinked polysaccharide colloidal particles formed according to the present invention by intramolecular and / or intermolecular crosslinking of linear polysaccharides, branched polysaccharides, or cyclic polysaccharides with a crosslinking agent may undergo chemical modifications that add, remove, or alter functional groups, such as -COOH, -NH2, and -OH groups, exposed on their surfaces. Such modifications may be performed to impart new chemical properties or enhance existing properties.

[251] Both chemical modifications and alterations, which refer to physical or structural changes in polysaccharide chains irrespective of the addition or removal of chemical functional groups, are important in the development of drugs or drug carriers based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention.

[252] The physical properties of non-immunogenic crosslinked polysaccharide colloidal particles, such as solubility or viscosity, may be optimized for a particular application by controlling the type and / or molecular weight and / or crosslinking density of the linear polysaccharide, branched polysaccharide, or cyclic polysaccharide. Furthermore, chemical modifications may be directed toward introducing new functionality or enhancing specific chemical interactions.

[253] For the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, the hydrodynamic diameter and surface charge may be precisely adjusted as desired by controlling at least one of the molecular weight of the polysaccharide, the length of the polysaccharide backbone, the type of crosslinking agent used for crosslinking, the amount and addition rate of the crosslinking agent during the synthesis reaction, and additional chemical functional group modification. This may ultimately provide the desired blood circulation time and the desired pharmacokinetics of biodistribution and elimination.

[254] In the present invention, a crosslinker-derived functional group exposed on the surface of a non-immunogenic crosslinked polysaccharide colloidal particle may be a terminal functional group of the crosslinking agent itself or a modified / substituted functional group thereof. For example, the crosslinker-derived functional groups exposed on the surface of the crosslinked polysaccharide colloidal particle may result from modification / substitution of at least some of the surface-exposed functional groups of the crosslinking agent.

[255] In the present invention, hydrophilic functional groups may be derived from functional groups of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide that did not participate in the crosslinking reaction; functional groups at one terminus of a crosslinking agent that did not participate in the crosslinking reaction; and / or functional groups obtained by further modification of one terminus of a crosslinking agent exposed after the crosslinking reaction.

[256] Non-limiting examples of crosslinker-derived functional groups or hydrophilic functional groups exposed on the surfaces of non-immunogenic crosslinked polysaccharide colloidal particles include amino groups, carboxyl groups, hydroxyl groups, and / or thiol groups. Reactive functional groups, such as amino, thiol, carboxyl, and hydroxyl groups, facilitate not only surface modification but also chemical conjugation with biopharmaceuticals, such as ligands that specifically bind to receptors on particular cells, antibodies or fragments thereof, antigenic peptides, and nucleic acids (DNA, RNA, or fragments thereof, etc.), or with various types of small-molecule drugs. In addition, in some cases, degradation in the acidic environment surrounding a tumor (pH < 7) or by hydrolases may release the drug in an active form. For example, non-limiting examples of acid-sensitive linkages that are degraded in the acidic environment surrounding a tumor (pH < 7) include carbonate or ester linkages.

[257] When surface-exposed functional groups of the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention carry a positive charge, as in the case of amino groups, cytotoxicity or the like may occur, as with other cationic polymers. This may be addressed by replacing some or all of the amino groups with carboxyl groups, methyl groups, ethyl groups, or the like.

[258] Non-limiting examples of hydrophilic functional groups that coordinate with metal ions, such as iron ions, include amino groups, thiol groups, carboxyl groups (carboxylate groups and carboxylic acid groups), and hydroxyl groups.

[259] In addition, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are suitable for various chemical modifications, such as esterification, etherification, and grafting. Such modifications may greatly alter their behavior in various physiological environments.

[260] Accordingly, various crosslinked polysaccharide colloidal particle derivatives having properties specific to particular uses may be generated through chemical modification of functional groups, such as -COOH, -NH2, and -OH groups, exposed on the surfaces of the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, and these derivatives also fall within the scope of the present invention.

[261]

[262] [Non-immunogenic Crosslinked Polysaccharide Colloidal Particles Having Prolonged Residence Time at the Injection Site and Targeting Capability]

[263] The non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may be conjugated with drugs, including contrast agents, or used as drug carriers, and are useful as a means of maintaining drugs for therapeutic or diagnostic purposes locally at high concentrations at a target site. In particular, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are designed to have a hydrodynamic diameter of about 2-10 nm, preferably 8 nm or less, and more preferably 6 nm or less, and a surface charge adjusted to a range of -20 mV to 0 mV, such that they are filtered in the kidneys but do not pass through normal capillaries and are selectively drained only through lymphatic vessels.

[264] Owing to these properties, even when not targeted, the particles of the present invention do not rapidly diffuse into the vasculature at the injection site, and 50% or more of the administered dose may remain at the injection site for 1 hour or more, preferably 2 hours or more, before being drained into lymphatic vessels. This induces expansion of the interstitial space or distension of anatomical spaces, allowing the particles to function as fillers or spaceexpanding agents (Examples 4 to 7).

[265] The mechanism underlying the prolonged residence time is interpreted as being attributable to the greater molecular weight and size of the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention relative to single-molecule drugs and to their restricted diffusion within tissue structures such as the extracellular matrix (ECM). In fact, when a T1 MRI contrast agent based on the particles of the present invention was injected into a joint cavity, intradermally, or subcutaneously, it was confirmed to be drained exclusively into lymphatic vessels, with imaging maintained for up to 1-2 hours after injection (see Examples 4-6).

[266] In addition, as a localized drug delivery system, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may compensate for unfavorable pharmacokinetic / pharmacodynamic (PK / PD) properties that may arise from rapid systemic diffusion of a therapeutic agent and contribute to reducing adverse effects and improving therapeutic efficacy.

[267] Furthermore, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may be modified through surface amino groups or -COOH functional groups with ligands, receptor agonists, or receptor antagonists capable of binding to specific cellsurface receptors. In this case, the particles may selectively remain at the target site for 1 day or more, preferably 4 days or more, while sustaining local activity (Example 2-7). In particular, when conjugated with antibodies or small-molecule ligands, the particles may specifically bind to receptors expressed on target cells and subsequently be eliminated from the body via the lymphatic system, thereby providing both targeting capability and control of biodistribution.

[268] Accordingly, the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention constitute a technological platform providing multiple advantages, including prolonged residence time at the injection site, distension of anatomical structures, selective lymphatic drainage, cell targeting, suppression of systemic diffusion, and improved local diagnostic and therapeutic efficiency, and are highly suitable as diagnostic and therapeutic compositions or drug carriers.

[269]

[270] [Targeted Contrast Agents and Targeted Drugs / Carriers]

[271] The present invention provides a metal complex-based targeted contrast agent or drug carrier platform usable as a T1 or T2 contrast agent for magnetic resonance imaging (MRI), by coordinating iron ions (Fe2+ / Fe3+), gadolinium ions (Gd3+), manganese ions (Mn2+), or iron oxide particles to surface-exposed crosslinker-derived amino groups or -COOH functional groups of non-immunogenic crosslinked polysaccharide colloidal particles.

[272] The non-immunogenic crosslinked polysaccharide colloidal particles of the present invention are designed so that, in a non-targeted state, they do not bind nonspecifically to cellsurface receptors or undergo cellular uptake. Accordingly, by introducing antibodies, peptides, small-molecule ligands, or the like having high affinity for specific cell receptors onto the particle can cause the particle to function as a contrast agent or drug carrier that accumulates selectively in target cells or tissues.

[273] For example, a targeted contrast agent may specifically bind to a particular tissue or cell and shorten the T1 relaxation time, thereby making the corresponding region appear bright in MR images and allowing the distribution or concentration of a target biomarker to be inferred from the images. In particular, T1 signal enhancement enables detection of small biological changes or pathological signals and may provide higher sensitivity and specificity than conventional non-targeted contrast agents.

[274] In addition, because targeted contrast agents selectively accumulate at specific sites, their doses can be reduced, thereby reducing the likelihood of adverse effects. Furthermore, they enable functional and molecular imaging in addition to anatomical imaging and may thus provide improved accuracy and information for disease diagnosis and pathophysiological assessment.

[275] Representative applications include cancer detection, involving highly sensitive imaging of early tumors and metastatic lesions using antibody-based contrast agents that bind to cancer cell-specific markers, such as HER2 and EGFR; visualization of inflammation and infection, involving detection of infectious foci using contrast agents conjugated with ligands for receptors associated with inflammatory responses, such as TLRs and integrins, or for bacterial / viral surface antigens; and diagnosis of neurological diseases, involving improved precision of brain imaging using contrast agents specific for Alzheimer’s disease-associated structures, such as amyloid plaques and tau protein, or for neurotransmitter receptors.

[276] Accordingly, targeted contrast agents and drug carriers based on the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention constitute a highly functional imaging nanoplatform offering the potential for disease detection at the molecular level, early diagnosis, assessment of targeted therapy, and imaging, and are suitable for diagnosis of various diseases and monitoring of personalized treatment.

[277]

[278] [Uses of Non-immunogenic Crosslinked Polysaccharide Colloidal Particles]

[279] The non-immunogenic crosslinked polysaccharide colloidal particles of the present invention may be designed to form covalent or coordination bonds with proteins, peptides, oligonucleotides, small-molecule drugs, metal ions, or nanoparticles using surface-exposed crosslinker-derived amino groups or carboxyl groups (-COOH). Accordingly, the non-immunogenic particles of the present invention may be used as drug carriers capable of carrying various drugs or functional nanomaterials, or as contrast agents themselves.

[280] In particular, because the non-immunogenic particles of the present invention have a high surface-to-volume ratio, they have high drug-loading efficiency per unit mass, and the drug-to-particle ratio (DPR) may be set quantitatively and uniformly by controlling surface functional groups. This contributes to improved predictability and repeatability of the pharmacokinetic behavior of the drug carriers.

[281] By precisely controlling properties such as colloidal stability, surface charge, hydrodynamic diameter, compressibility, and immune evasion, the non-immunogenic particles of the present invention may have uses as drug carriers, including as colloidal stabilizers for injection into the body; agents for enhancing tissue permeability or suppressing outflow; agents for controlling surface charge and hydrodynamic diameter; surface-modifying agents for evading phagocytosis by macrophages; agents for controlling lymphatic or blood circulation half-life; agents for controlling drug biodistribution or elimination; contrast agents for location tracking by MRI; and nanoplatforms for drug loading and release.

[282] In addition, attachment of the non-immunogenic particles of the present invention to the surfaces of drugs or nanoparticles may provide effects such as minimizing immunogenicity, enabling repeat administration, avoiding antigen exposure, prolonging residence time in the blood, and reducing rapid immune clearance.

[283] The non-immunogenic particles of the present invention may form a hydration layer through their hydrophilic structure, thereby preventing adsorption of opsonin proteins and evading recognition by macrophages, which improves the stability and persistence of drugs or particles in vivo.

[284] Furthermore, the non-immunogenic particles of the present invention may improve the colloidal stability of drugs that are insoluble or poorly soluble in vivo and may therefore be designed as drug delivery systems optimized for various routes, such as intravenous, subcutaneous, intramuscular, or oral administration.

[285]

[286] [Non-immunogenic Crosslinked Polysaccharide Colloidal Particle-Drug Complexes]

[287] The present invention provides crosslinked polysaccharide colloidal particle-drug complexes (polysaccharide cross-linked particle-drug conjugates, PDCs) by forming covalent bonds, coordination bonds, hydrogen bonds, or electrostatic bonds with drugs in various forms, such as nanoparticles, small molecules, peptides, proteins, and nucleic acids, through surface-exposed amino groups or carboxyl groups (-COOH) of non-immunogenic crosslinked polysaccharide colloidal particles.

[288] A drug is a substance intended for the diagnosis, treatment, alleviation, or prevention of a disease, or for affecting physiological functions. In the present invention, drugs comprise biologically active substances, such as small-molecule compounds, peptides, proteins (e.g., antibodies), and oligonucleotides (RNA, aptamers). In particular, ultra-toxic payloads having IC50 values in the pM range, such as tubulin inhibitors, DNA-damaging agents, and Top-1 inhibitors, are also included.

[289] In the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention, the number of functional groups and the surface charge may be appropriately adjusted according to the type and structure of the drug. The particles are also capable of forming coordination bonds with metal ions, such as Fe2+ and Fe3+, or metal oxides, such as iron oxide nanoparticles, and may therefore be used as surface-modifying agents for functional inorganic particles.

[290] Specific examples of the complexes include forms in which a payload is conjugated through a degradable linker, forms in which the particles are attached as modifying agents to the surfaces of aggregated hydrophobic drug nanoparticles, and electrostatic complexes of negatively charged nucleic acid-based drugs and positively charged crosslinked polysaccharide colloidal particles.

[291] In addition, the non-immunogenic crosslinked polysaccharide colloidal particle-drug complexes of the present invention have in vivo functions such as imparting colloidal stability (maintaining stability in body fluids through formation of a hydration layer by hydrophilic functional groups), controlling pharmacokinetics (enabling control of blood circulation time and renal or hepatic clearance by adjusting hydrodynamic diameter and surface charge), immune evasion (suppressing phagocytosis by phagocytes and antibody formation through a non-immunogenic coating), and targeted drug release (enabling selective drug release in environments such as the tumor microenvironment when acid-sensitive linkers are used, as a prodrug strategy).

[292] When the surfaces of functional nanoparticles, such as superparamagnetic iron oxide, gold, and quantum dots, are coated with the non-immunogenic particles of the present invention, their hydrodynamic diameter, surface charge, and biodistribution are controlled, and evasion of phagocytosis and prolonged residence time in the bloodstream are expected. This increases their potential use for bioimaging, drug delivery, and reusable platforms.

[293] In particular, the drug-to-particle ratio (DPR) of the PDCs according to the present invention may be precisely controlled, which is critical to drug delivery efficiency and aggregation stability. The DPR may be adjusted within a range of 1-10, preferably 1-8, more preferably 1-6, and still more preferably 1-4, and a range of 1-3 is more suitable for hydrophobic drugs or peptides for which immunogenicity is a concern. Uniformity of the DPR contributes to improved pharmacokinetic predictability and repeatability of administration of the drug complexes.

[294] In conclusion, the non-immunogenic crosslinked polysaccharide colloidal particle-drug complexes of the present invention have technical advantages such as applicability to various drugs and functional nanoparticles (versatility), control of biodistribution and elimination (predictability), provision of colloidal stability (physicochemical stability), suppression of immune responses and enablement of repeat administration (clinical safety), and targeted drug release at tumor or infection sites (therapeutic efficiency). Advantageous Effects of the Invention

[295] The non-immunogenic crosslinked polysaccharide colloidal particles according to the present invention have precisely designed surface structures so that they are not recognized by immune cells or do not activate immune cells, thereby minimizing immune responses upon injection into the body. The particles of the present invention are not readily degraded by enzymes in the body and may be stably eliminated from the body without hypersensitivity reactions even in pathological environments with immune activation, such as inflammatory lesions.

[296] In addition, even when various polysaccharide polymers are used as polysaccharides to be cross-linked, structural heterogeneity that may arise in the three-dimensional network structure formed through polysaccharide crosslinking can be controlled by selectively modifying the -OH functional groups of monosaccharides with an epoxide-based first crosslinking agent and controlling chemical crosslinking, crosslinking conditions, and surface modification. This may suppress nonspecific interactions with immune cells and reduce the likelihood of adverse effects arising from interspecies differences in immune recognition or metabolic pathways.

[297] The surface charge of the non-immunogenic particles of the present invention may be precisely adjusted within a range of -20 mV to 0 mV, enabling evasion of phagocytosis by macrophages, prolonged circulation time, and control of elimination from the body. When the particles are used as drug delivery platforms, contrast agents, or nanoparticles for targeted diagnosis, these properties contribute to improved drug efficacy and reduced dose and dosing frequency by slowing drug elimination and maintaining blood concentrations.

[298] In particular, the crosslinked polysaccharide colloidal particles according to the present invention act as non-immunostimulatory nanoparticles and may function as drugs themselves, drug carriers, or stealth coating agents. Accordingly, adverse effects associated with immune activation are suppressed, and the particles may be used even upon repeat administration without immune tolerance.

[299] The platform of the present invention also has substantial potential for application as a T1 MRI contrast agent. It is designed to be drained exclusively into lymphatic vessels upon intradermal or subcutaneous administration, enabling selective visualization of only peripheral lymphatic vessels without venous contamination. This is suitable for various clinical applications, such as MR lymphangiography and postoperative tumor imaging, and may provide morphological and functional information useful for planning microsurgical procedures, such as lymphaticovenous anastomosis (LVA). Because it is also drained exclusively into lymphatic vessels upon intra-articular administration, it remains in the joint cavity longer than conventional small-molecule contrast agents that are drained into both lymphatic vessels and blood vessels, making it suitable for MR arthrography and similar applications.

[300] The present invention provides key effects such as minimizing immune responses and enabling repeat administration; prolonging the circulation time of drugs / contrast agents in the body and precisely controlling their elimination; providing resistance to enzymatic degradation in the body and stability in inflammatory environments; application as a platform technology for precise diagnostic imaging and lesion-targeted delivery; and enhancing industrial utility in various diagnostic and therapeutic fields. Brief Description of the Drawings

[301] Hereinafter, the crosslinked dextran structure (nanoparticle) is referred to interchangeably as CDex, DNP, and C-DNP.

[302] FIG. 1 shows schematic structures of INV-001 and NEMO-103 Injection, methods for preparing the same, and their physicochemical properties.

[303] FIG. 2 compares the in vivo acute edema response to NEMO-103 prepared using crosslinked dextran (Cdex) with that to Dextran T10.

[304] FIG. 3 shows the conjugation chemistry processes for Cdex and a Toll-like receptor (TLR) agonist (a), the chemical structures of Cdex@TLR obtained using the respective conjugation chemistry methods (b), and the ratios of TLR agonist conjugated per Cdex using the respective conjugation chemistry methods (c).

[305] FIG. 4 shows immunofluorescence staining images of the NF-kB p65 signal after 2 hours of treatment in Example 2-5.

[306] FIG. 5 shows immunofluorescence staining images of the NF-kB p65 signal after treatment of cells with a TLR agonist for 4 hours and 6 hours, respectively.

[307] FIG. 6 shows cell viability as a function of concentration, determined using a cell counting kit-8 (CCK-8) after treatment of cells with Cdex, a TLRA1agonist, Cdex@TLRA1, and Cdex@TLRB1for 4 hours.

[308] FIG. 7 shows the experimental design and results of an in vivo efficacy evaluation following administration of Doxorubicin, a TLRA1 agonist, Cdex@TLRA1 , Cdex@TLRA1X2 (twice the dose), and Cdex@TLRB1to xenograft model mice, with a control group.

[309] FIG. 8 shows structural formulas of examples of branched polysaccharides or cyclic polysaccharides to be cross-linked to form the crosslinked polysaccharide colloidal particles in Example 3.

[310] FIG. 9 shows T1-weighted MR images acquired after intravenous administration to mice of the materials synthesized in Example 3-11 by introducing iron into crosslinked Dextran T-5, Maltodextrin, a-cyclodextrin, P-cyclodextrin, and Inulin (a), and the signal-to-noise ratios (SNRs) before and after administration (b).

[311] FIG. 10 shows T1-weighted MR images confirming renal excretion after intravenous administration to mice of the materials synthesized in Example 3-12 by introducing iron into crosslinked Dextran T-5, Maltodextrin, a-cyclodextrin, P-cyclodextrin, and Inulin.

[312] FIG. 11 shows MR arthrography (MRA) images acquired 30 minutes (a) and 60 minutes (b) after administration of NEMO-103 Injection to humans in the Phase 1 / 2a clinical trial of NEMO-103 Injection, and a comparison of these images in terms of CNR (c), Distension (d), and Overall quality (e).

[313] FIG. 12 shows MRA images acquired approximately 30 minutes after administration of NEMO-103 Injection (a) and a gadolinium-based contrast agent (GBCA) (b) to humans, and a comparison of these images in terms of contrast-to-noise ratio (CNR) (c), Distension (d), and Overall quality (e).

[314] FIG. 13 shows MRA images acquired 60 minutes after administration of NEMO-103 Injection (a, c) and GBCA (b, e) to humans, and a comparison with GBCA in terms of CNR (c), Distension (d), and Overall quality (e).

[315] FIG. 14 shows the results of assessments of joint distension, joint boundary definition, and contrast before and after administration of NEMO-103 Injection to humans in the Phase 2b clinical trial of NEMO-103 Injection.

[316] FIG. 15 compares the effects of Gd-DOTA and INV-001 on contrast enhancement of the popliteal lymph node (LN) and lymphatic vessels in Example 5. (a) 3D-TOF scan images acquired before (0 minutes), and 16 minutes and 96 minutes after, intradermal injection of Gd-DOTA and INV-001. (b) Analyses of the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the popliteal LN and lymphatic vessel (LV) after administration of Gd-DOTA and INV-001.

[317] FIG. 16 shows the results of a dose optimization study conducted in Example 5 to minimize improvement of the interstitial space and lymph node enlargement. The popliteal LN and LV were visualized 16 minutes after injection into the hindlimb at various concentrations and doses.

[318] FIG. 17 shows lymphatic vessel images obtained by magnetic resonance lymphangiography (MRL) after injection of INV-001 and Gd-based contrast agents in various animal models (rats, minipigs, and beagle dogs). Each panel compares the degree of lymphatic vessel visualization and the presence or absence of venous contamination after contrast agent injection. The images show that INV-001 maintains lymphatic vessel contrast enhancement relative to Gd contrast agents without venous contamination, and that its efficacy is maintained even in a disease model involving lymph node resection.

[319] FIG. 18 shows images in which INV-001 injected into the distal hindlimb of a beagle dog provides contrast enhancement extending to the central lymphatic system.

[320] FIG. 19 shows magnetic resonance lymphangiography (MRL) images obtained after injection of INV-001 into humans.

[321] FIG. 20 shows representative INV-MRL and NIRF-ICGL images in dorsal (D) and ventral (V) views. In the control limb, the popliteal lymph node (LN) was observed with both modalities (red arrows). The orange arrows indicate regions showing a diffuse pattern in NIRF-ICGL, in which the contrast agent is widely distributed over the skin surface, making it difficult to observe the underlying lymphatic vessels (LVs). However, INV-MRL allows visualization of the obscured LVs at the same locations. Mode for Carrying Out the Invention

[322] Hereinafter, the present invention is described in greater detail through examples. However, the following examples serve only to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

[323] Hereinafter, the crosslinked dextran structure (nanoparticle) is referred to interchangeably as CDex, DNP, and C-DNP.

[324] Example 1. Pharmacokinetics of functional nanoparticles coated with crosslinked polysaccharide colloidal particles having controlled size and surface charge

[325] 1-1. Synthesis of C-DNP-3 by crosslinking dextran having a molecular weight of 3 kDa

[326] Dextran (molecular weight: 3 kDa; 1 g) was dissolved in 4.2 mL of distilled water, and 8.3 mL of a NaOH solution was added. Epichlorohydrin (3.3 mL) was added, and the mixture was stirred. Diethylenetriamine (DETA; 7 mL) was then added, and the mixture was stirred for an additional 24 hours to synthesize C-DNP-3 in which dextran having a molecular weight of 3 kDa was crosslinked. After purification by ultrafiltration, the hydrodynamic size measured by DLS was 3 nm.

[327] 1-2. Synthesis of C-DNP-5 by crosslinking dextran having a molecular weight of 5 kDa

[328] C-DNP-5 in which dextran having a molecular weight of 5 kDa was crosslinked was synthesized by the same method as in Example 1-1, except that dextran (molecular weight: 5 kDa) was used instead of dextran (molecular weight: 3 kDa). After purification by ultrafiltration, the hydrodynamic size measured by dynamic light scattering (DLS) was 4 nm.

[329] 1-3. Synthesis of C-DNP-10 by crosslinking dextran having a molecular weight of 10 kDa

[330] C-DNP-10 in which dextran having a molecular weight of 10 kDa was crosslinked was synthesized by the same method as in Example 1-1, except that dextran (molecular weight: 10 kDa) was used instead of dextran (molecular weight: 3 kDa). After purification by ultrafiltration, the hydrodynamic size measured by DLS was 5 nm.

[331] 1-4. Synthesis of C-CMDNP-10 by crosslinking carboxymethyl dextran (CM dextran) having a molecular weight of 10 kDa

[332] C-CMDNP-10 in which CM dextran having a molecular weight of 10 kDa was crosslinked was synthesized by the same method as in Example 1-1, except that CM dextran (molecular weight: 10 kDa) was used instead of dextran (molecular weight: 3 kDa). After purification by ultrafiltration, the hydrodynamic size measured by DLS was 5 nm.

[333] 1-5. Substitution of functional groups exposed on the surface of C-DNP or C-CMDNP

[334] 1-5-1. Amine group:

[335] The functional groups exposed on the surface of C-DNP or cross-linked CM dextran nanoparticles (C-CMDNP) synthesized in Examples 1-1 to 1-4 are amino groups.

[336] 1-5-2. Carboxyl group:

[337] Succinyl anhydride (SA; 30 mg) was added to 10 mL of C-DNP or C-CMDNP synthesized in Examples 1-1 to 1-4, and the mixture was stirred for 12 hours. The resulting C-DNP or C-CMDNP was purified by ultrafiltration.

[338] 1-5-3. Thiol group:

[339] N-Succinimidyl S-acetylthioacetate was added to 10 mL of C-DNP or C-CMDNP synthesized in Examples 1-1 to 1-4, and the mixture was stirred for 12 hours. The resulting C-DNP or C-CMDNP was purified by ultrafiltration.

[340] 1-5-4. Hydroxide group:

[341] Nitrous acid was added to 10 mL of C-DNP or C-CMDNP synthesized in Examples 1-1 to 1-4, and the mixture was stirred for 12 hours. The resulting C-DNP or C-CMDNP was purified by ultrafiltration.

[342] 1-6. Control of the surface charge of C-DNP / C-CMDNP

[343] When 30 mg of SA is added according to Example 1-5-2 to 10 mL of C-DNP synthesized in Examples 1-1 to 1-3, the surface charge is -3 mV. When the amount of SA added is 50 mg, the surface charge is -20 mV. When no SA is added, the surface charge is +5 mV. In experiments using C-DNP-3, C-DNP-5, and C-DNP-10, the surface charges as a function of the amount of SA added showed the same results under the same functional group substitution conditions. When 30 mg of SA is added according to Example 1-5-2 to 10 mL of C-CMDNP-10 synthesized in Example 1-4 and having a surface charge of +5 mV, the surface charge is -4 mV. Thus, the surface charge becomes more negative as the amount of SA added increases, allowing the surface charge to be controlled.

[344] 1-7. Introduction of fluorescent molecules into C-DNP and control of the number of molecules introduced

[345] 5 / 6-Carboxyfluorescein succinimidyl ester (NHS-fluorescein; 1 mg) was dissolved in 1 mL of dimethyl sulfoxide (DMSO). This solution, in volumes of 5, 10, and 25 pL, was added to 1 mg of C-DNP-5 having exposed amino groups (surface charge: +5 mV) from Example 15-1. After 24 hours, the C-DNP-5 into which fluorescein had been introduced was purified by ultrafiltration. The number of fluorescein molecules bound to C-DNP-5 reacted with 5 mmol of NHS-fluorescein / DMSO was 4, the number bound to C-DNP-5 reacted with 10 mmol of NHS-fluorescein / DMSO was 9, and the number bound to C-DNP-5 reacted with 25 mmol of NHS-fluorescein / DMSO was 15. As described above, the amount of a substance to be bound to C-DNP can be controlled by adjusting the reaction ratio.

[346]

[347] Example 2. Crosslinked polysaccharide colloidal particle-drug conjugate (Cdex@TLR as an anticancer agent)

[348] The mechanism of cancer cell killing by TLR agonists is described in detail in the literature (Remaut et al., Eur. J. Pharm. Biopharm., 2022, 172, 16).

[349] TLR agonists are known to activate various immune cells, including dendritic cells (DCs), macrophages, and T cells. This activation may enhance the body's immune response against cancer cells.

[350] 2-1. Preparation of a nanostructure (Cdex) using dextran T-10

[351] Dextran T-10 (average molecular weight: 10,000 Da; 180 pmol) was dissolved in 9 mL of distilled water, and 75 mmol of epichlorohydrin and 75 mmol of NaOH were added. Then, 380 mmol of ethylenediamine was added, and the mixture was stirred at room temperature (RT) for 24 hours. Succinic anhydride (25 mg) was added to this material at room temperature, and, after a succinylation reaction for 24 hours, the material was purified by dialysis using a 10 kDa molecular weight cutoff (MWCO) filter. The hydrodynamic size measured by DLS was 5 nm.

[352] An edema test was conducted following administration of NEMO-103 prepared using Cdex, the crosslinked form of dextran T-10, in comparison with dextran T-10 used as the starting material for crosslinking. When dextran and Cdex were intravenously administered to rats at the same doses (125 and 250 mg / kg), edema of the face and paws was observed after administration only in the case of dextran (FIG. 2).

[353] 2-2. Introduction of drugs (TLR 7 / 8 agonists) capable of binding to Cdex

[354] Various types of TLR 7 agonists are currently commercially available, and substances comprising an amino group capable of coupling with the crosslinker-derived amino groups exposed on the surface of Cdex of Example 2-1 were selected. Among these substances, two TLR 7 agonists (TLRA1 and TLRB1agonists) were selected in which the introduced amino group did not interfere with the chemical site required for binding to TLR 7.

[355] As illustrated in FIG. 3, Cdex and a TLR agonist were conjugated using chemical coupling methods employing EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-hydroxysuccinimide)-PEG (polyethylene glycol)-NHS, and BCN (bicyclo[6.1.0]nonyne)-NHS / N3 (azide)-NHS (Cdex@TLR, FIG. 3, a and b). For chemical coupling using N3-PEG-NHS, 30 mg of N3-PEG-NHS was dissolved in 2 mL of DMSO and added to the Cdex material of Example 2-1, followed by stirring at room temperature for 2 hours. BCN-NHS (10 mg) and the TLRA1agonist (10 mg) were mixed, dissolved in 2 mL of DMSO, and then added. After stirring for 2 hours, the material was purified by dialysis using a 10 kDa molecular weight cutoff (MWCO) filter. For chemical coupling using NHS-PEG-NHS, 10 mg of the TLRA1agonist and 20 mg of NHS-PEG-NHS were added to the Cdex material. After stirring for 2 hours, the material was purified by dialysis using a 10 kDa molecular weight cutoff (MWCO) filter.

[356] As a result, the number of TLRA1agonist molecules bound to Cdex was 3 for NHS-BCN / N3-NHS chemical coupling and 0.45 for NHS-PEG-NHS chemical coupling, confirming 67 that the NHS-BCN / N3-NHS chemical coupling method was the most efficient in terms of chemical coupling efficiency (FIG. 3, c).

[357] 2-3. Quantification of the drug bound to Cdex

[358] The material of Example 2-2 was diluted to 0.5 ^g / mL and placed in a cuvette, and its absorbance was measured at wavelengths of 200-500 nm using a UV-Vis spectrophotometer. Cdex without a bound drug showed no absorption at 350 nm, whereas Cdex with a bound drug showed absorption at 350 nm. This measurement was used to quantify the amount of drug bound to Cdex and determine the number of TLRA1agonist molecules bound per mole of Cdex. It was confirmed that three or more TLRA1agonist molecules were bound per Cdex.

[359] 2-4. Hydrodynamic diameter and colloidal stability of Cdex@TLR

[360] Cdex conjugated with the TLRA1 agonist in Example 2-2 was confirmed to have a hydrodynamic diameter (hydrodynamic size) of 6 nm and to remain stably dispersed without aggregation even in a 1 M NaCl solution, a condition much harsher than normal physiological conditions.

[361] 2-5. Evaluation of the in vitro efficacy of Cdex@TLR (assessment of the NF-kB p65 signal)

[362] The efficacy of the crosslinked polysaccharide colloidal particles of the present invention as drug carriers can be evaluated by examining whether treatment with Cdex@TLRA1 or Cdex@TLRB1induces the TLR signaling pathway (NF-kB signaling) in cells expressing TLRs (Toll-like receptors).

[363] To determine whether the NF-kB signaling pathway was induced through activation of TLR signaling, the presence or absence of TLR signaling activation following treatment with Cdex@TLR of Example 2-2 was examined using the RAW 264.7 macrophage cell line, a representative immune cell line.

[364] Specifically, cells of the mouse RAW 264.7 macrophage cell line were treated with the two samples prepared in Example 2-2, Cdex@TLRA1 and Cdex@TLRB1 , and the controls, Cdex and TLR 7 / 8 agonists (TLRA1 and TLRB1 agonists). Observations were made by immunofluorescence staining for NF-kB p65 at the treatment concentrations and times presented in Table 2.

[365] [Table 2] Treatment group Sample name Treatment concentration (pM) Treatment time (h) Detection Control Untreated - 2, 4, 6 Immunofluorescent Cdex 2.0, 3.0 TLR 7 / 8 Agonist TLRa1 6.0, 9.0 TLRb1 Cdex@TLR Cdex@TLRA1-3 Cdex@TLRB1-3

[366] Observation by NF-kB p65 immunofluorescence staining after 2 hours of treatment showed that, as illustrated in FIG. 4, fluorescence was observed throughout the cytoplasm in the controls (cell only and Cdex), whereas stronger NF-kB p65 fluorescence was observed in the nucleus than in the cytoplasm for the TLR 7 / 8 agonists ( TLRai and TLRbi agonists), Cdex@TLRA1, and Cdex@TLRB1. It is known that activation of the TLR signaling pathway results in a stronger NF-kB p65 fluorescence signal in the nucleus than in the cytoplasm. Since Cdex@TLRA1 and Cdex@TLRB1 exhibited relatively greater NF-kB p65 fluorescence intensities than the TLRA1 and TLRB1 agonists, Cdex@TLRA1 and Cdex@TLRB1 were confirmed to be effective in activating TLR signaling. Comparison of fluorescence intensities at different treatment concentrations showed that the NF-kB p65 fluorescence signal intensity at 6 pM was similar to that observed after treatment at 9 pM.

[367] An optimal agonist was selected from various TLR 7 / 8 agonists by examining the level of NF-kB p65 expression observed upon treatment of cells. Mouse RAW 264.7 macrophage cells were treated with Gardiquimod, Resiquimod, the TLRA1agonist, and the TLRB1agonist at 6 pM for 4 hours. After 4 hours, each cell lysate was separated into cytoplasmic and nuclear fractions, and the NF-kB p65 signal was evaluated by Western blotting. Lamin (nuclear) and P-actin (cytoplasmic) were used as housekeeping proteins for Western blot normalization, respectively. Comparison of the four agonists showed that the TLRA1 agonist produced the highest nuclear NF-kB p65 expression. Treatment with Resiquimod and the TLRB1 agonist showed relatively lower NF-kB p65 expression than treatment of cells with the same volume of DMSO.

[368] In a similar experiment, mouse RAW 264.7 macrophage cells were treated with Gardiquimod, Resiquimod, the TLRA1agonist, and the TLRB1agonist at 6 pM for 4 hours and 69 6 hours, after which NF-kB p65 expression was observed by immunofluorescence staining (FIG. 5). Treatment with the TLRA1agonist for 4 hours showed the highest nuclear NF-kB p65 expression, confirming that it had the greatest effect on TLR signaling activation.

[369] In summary, FIG. 4 suggests that the crosslinked polysaccharide colloidal particles of the present invention, as drug carriers, provide an orientation in which small-molecule drugs, such as TLR 7 / 8 agonists, uniformly distributed on their surface increase avidity for cell surface receptors (TLRs) without aggregation.

[370] 2-6. Evaluation of cell viability in TLR-expressing immune cells treated with Cdex@TLR

[371] Cell viability was tested at different treatment concentrations and times using the two ADC platforms whose efficacy had been confirmed, Cdex@TLRA1and Cdex@TLRB1, as well as Cdex and TLR 7 / 8 agonists (TLRA1 and TLRB1agonists). The evaluation was performed using the TLR-expressing mouse RAW 264.7 macrophage cell line.

[372] Specifically, cells were assigned to an untreated group and groups treated with the ADC platform at low, medium, and high concentrations. Changes in the cells according to treatment concentration and time were examined using the CCK-8 assay, a cell viability assay.

[373] The results of the CCK-8 assay after 4 hours of treatment confirmed that Cdex itself caused no change in cell viability with treatment concentration. For the TLRA1 agonist, cell viability was confirmed to decrease to approximately 89% starting at 30 pM. In the case of Cdex@TLRA1 and Cdex@TLRB1, cell viability was enhanced compared with the untreated group through stimulation of the NF-kB activation pathway (FIG. 6). These results indicate that, whereas the TLRA1 agonist aggregated at high concentrations, Cdex@TLRA1 and Cdex@TLRB1 activate TLR-expressing immune cells without aggregation even at high concentrations.

[374] 2-7. Confirmation of drug efficacy in cancer xenograft mice—in vivo efficacy

[375] Drug efficacy was confirmed by monitoring changes in tumor size following drug injection. Cancer cell killing resulting from administration of Cdex@TLRA1 and Cdex@TLRB1and the consequent immune response was confirmed in cancer xenograft mice.

[376] Balb / c nude mice (6 weeks old) were used as experimental animals, and a BT-474 breast cancer xenograft mouse model was established to evaluate in vivo efficacy. A mixture of 1 x 106 BT-474 breast cancer cells and Matrigel at a 1:1 volume ratio was implanted subcutaneously into the dorsal region of the mice in a volume of approximately 100 pL.

[377] When the tumor size reached approximately 50 mm3, test substances including Cdex@TLRA1and Cdex@TLRB1 were administered directly into the tumors, and drug efficacy was confirmed by measuring changes in tumor size for 2 weeks. The test substances were administered on day 0, day 4, day 7, day 12, and day 15 (FIG. 7). For effective cancer cell killing, Doxorubicin was administered on Day 1 and Day 9 to all groups except the control group, and a separate Doxorubicin-only group was established for comparison (FIG. 7 and Table 3).

[378] [Table 3] Group Sample name TLR / Cdex Administration concentration Administration volume (^L) Number of animals 1 Control (1XPBS) - 20 3 2 Doxorubicin (Dox) only 2 mg / kg 20 4 3 TLRA1agonist 6 nmol / mL (0.3 nmol) 20 3 4 Cdex@TLRA1 2.4 6 nmol / mL (0.3 nmol) 20 3 5 Cdex@TLRA1x2 2.4 12 nmol / mL (0.6 nmol) 20 4 6 Cdex@TLRB1 2.8 6 nmol / mL (0.3 nmol) 20 3

[379] Relative tumor volume was observed to decrease over time in the groups treated with Cdex@TLRA1 , Cdex@TLRA1 x2, and Cdex@TLRB1 , compared with the control group (injected with phosphate-buffered saline, PBS) and the groups treated with Doxorubicin and TLRa1.

[380] An anticancer therapeutic effect was confirmed by observing a decrease in relative tumor volume over time in the groups treated with Cdex@TLRA1 , Cdex@TLRA1 x2, and Cdex@TLRB1, compared with the control group (injected with PBS). In the Doxorubicin-only group and the TLRA1 agonist-treated group, tumor growth was observed to slow, but no reduction in tumor size was observed.

[381] In summary, these results suggest that, at tumor sites in vivo, the crosslinked polysaccharide colloidal particles of the present invention, as drug carriers, activated various immune cells, including dendritic cells (DCs), macrophages, and T cells, which can enhance the body's immune response against cancer cells, by providing an orientation in which the TLR agonists uniformly distributed on their surface increase avidity for cell surface receptors (TLRs) on immune cells without aggregation.

[382]

[383] Example 3. T1 MRI contrast agents based on the crosslinked polysaccharide colloidal particles of the present invention

[384] In this example, a study was conducted to form crosslinked polysaccharide colloidal particles by crosslinking various polysaccharides, including dextran (FIG. 8), and to determine whether they could be used as T1 MRI contrast agents.

[385] 3-1. Formation of a nanostructure using dextran T-5

[386]

[387] Dextran T-5 (FIG. 8(a-i), average molecular weight: 5,000 Da; 180 pmol) was dissolved in 9 mL of distilled water, and 75 mmol of epichlorohydrin and 75 mmol of NaOH were added. Then, 380 mmol of diethylenetriamine was added, and the mixture was stirred at room temperature (RT) for 24 hours, followed by purification using a 5 kD molecular weight cutoff (MWCO) filter.

[388] 3-2. Synthesis of a nanostructure using maltodextrin

[389] All experiments were performed in the same manner as in Example 3-1, except that maltodextrin (FIG. 8(a-ii), average molecular weight: 990 Da) was used instead of dextran T-5.

[390] 3-3. Synthesis of a nanostructure using alpha-cyclodextrin

[391] All experiments were performed in the same manner as in Example 3-1, except that alpha-cyclodextrin (FIG. 8(b-i), average molecular weight: 970 Da) was used instead of dextran T-5.

[392] 3-4. Synthesis of a nanostructure using beta-cyclodextrin

[393] All experiments were performed in the same manner as in Example 3-1, except that betacyclodextrin (FIG. 8(b-ii), average molecular weight: 1100 Da) was used instead of dextran T-5.

[394] 3-5. Synthesis of a nanostructure using inulin

[395] All experiments were performed in the same manner as in Example 3-1, except that inulin (FIG. 8(c), average molecular weight: 2500 Da) was used instead of dextran T-5.

[396] To confirm crosslinking of the materials of Examples 3-1 to 3-5, the materials were prepared at the same concentration and placed in cuvettes, and their absorbance was measured at wavelengths of 200-400 nm using a UV-Vis spectrophotometer. Whereas ordinary polymers exhibit no absorption in the visible region, nanoparticles formed after crosslinking scatter transmitted light, which is consequently observed as absorption at short wavelengths. Before crosslinking, no absorption by the polymers was observed in the measured wavelength region, whereas the materials in which nanostructures had formed exhibited absorption in the shortwavelength region (200-250 nm), confirming the formation of nanostructures through crosslinking.

[397] Amino groups are formed on the surfaces of the nanostructures synthesized in Examples 3-1 to 3-5 by diethylenetriamine. Analysis using an o-phthalaldehyde assay to quantify the number of surface amino groups per nanostructure showed 12.7 for dextran T-5, 4.5 for maltodextrin, 8.1 for alpha-cyclodextrin, 9.0 for beta-cyclodextrin, and 8.0 for inulin. Thus, it was confirmed that this synthesis method introduces amino groups onto the surfaces of all the nanostructures.

[398] 3-6. Introduction of carboxyl groups into the nanostructures

[399] Succinic anhydride (25 mg) was added to the materials of Examples 3-1 to 3-5 at room temperature. After a succinylation reaction for 24 hours, the materials were purified using a 5 kD molecular weight cutoff (MWCO) filter.

[400] The number of residual amino groups in the materials of Example 3-6 was determined using an o-phthalaldehyde assay. The number of residual amino groups per nanostructure was determined to be 0.7 for dextran T-5, 0.2 for maltodextrin, 0.2 for alpha-cyclodextrin, 0.2 for beta-cyclodextrin, and 0.4 for inulin. A residual amino group count of 1 or less means that all amino groups have been replaced with carboxyl groups. Accordingly, in this reaction, all amino groups of the nanostructures prepared using the various polymers were replaced with carboxyl groups.

[401] 3-7. Introduction of iron into the nanostructures

[402] An iron chloride hexahydrate solution (45 pL) was added to the materials of Example 36. After the pH was adjusted to 8 using 2.5 M NaOH, the materials were reacted at room temperature for 1 hour and then purified using a 5 kD molecular weight cutoff (MWCO) filter to synthesize the nanostructures.

[403] The iron content and polymer content of the materials of Example 3-7 were analyzed. Surface-bound iron was analyzed by inductively coupled plasma-optical emission spectroscopy (ICP-OES), and the content of each polymer was analyzed by the phenol-sulfuric acid method. The amounts of iron bound to 1 mg of polymer were determined to be 0.03 mg for dextran T-5, 0.026 mg for maltodextrin, 0.033 mg for alpha-cyclodextrin, 0.026 mg for beta-cyclodextrin, and 0.029 mg for inulin. This reaction confirmed that similar amounts of iron were introduced when the iron introduction reaction was performed using various polymers.

[404] 3-8. Measurement of the hydrodynamic diameter and surface charge of the iron-containing nanostructures

[405] Analysis of the hydrodynamic diameters of the materials of Example 3-7 using DLS showed 3.6 nm for dextran T-5, 6.8 nm for maltodextrin, 2.9 nm for alpha-cyclodextrin, 2.8 nm for beta-cyclodextrin, and 3.8 nm for inulin, confirming that the materials had similar hydrodynamic diameters. The surface charges were determined to be -3.01 mV for dextran T-5, -6.62 mV for maltodextrin, -9.06 mV for alpha-cyclodextrin, -7.32 mV for betacyclodextrin, and -2.83 mV for inulin. Thus, it was confirmed that the iron-containing nanostructures formed using this synthesis method had similar hydrodynamic diameters and surface charges.

[406] 3-9. Measurement and comparison of the viscosities of the nanostructures

[407] The materials of Example 3-7 were prepared at a concentration of 5 mg / mL, and their viscosities were measured at 25°C. The viscosities of the crosslinked polymers were similar, with values of 1.09 mPa^s for dextran T-5, 1.43 mPa^s for maltodextrin, 1.26 mPa^s for alphacyclodextrin, 1.3 mPa^s for beta-cyclodextrin, and 1.14 mPa^s for inulin.

[408] 3-10. Measurement and comparison of the T1 MRI contrast enhancement effects of the nanostructures

[409] To analyze the T1 MRI performance of the materials of Example 3-7, the spin-spin relaxivity coefficient (n) and spin-lattice relaxivity coefficient (ri) were measured, and their ratio (r2 / ri ratio) was calculated. The r2 / ri ratio serves as an index for determining whether a contrast agent is suitable as a T1 MRI contrast agent or a T2 MRI contrast agent. Analysis of the materials of Example 3-7 by 3.0 Tesla MRI showed the following results: dextran, ri of 1.90, r2 of 2.27, and an r2 / ri ratio of 1.19; maltodextrin, ri of 4.60, r2 of 5.19, and an r2 / ri ratio of 1.13; alpha-cyclodextrin, ri of 5.26, r2 of 5.78, and an r2 / ri ratio of 1.10; beta-cyclodextrin, ri of 5.62, r2 of 6.24, and an r2 / ri ratio of 1.11; and inulin, ri of 4.33, r2 of 4.73, and an r2 / ri ratio of 1.09. Thus, all the synthesized materials were confirmed to exhibit T1 MRI contrast enhancement effects because their r2 / ri ratios were close to 1.

[410] 3-11. Evaluation of the contrast enhancement efficacy of the iron-containing nanostructures in animals

[411] T1-weighted images were acquired by 9.4 Tesla MRI after intravenous administration of the materials of Example 3-7 to mice. Male Balb / c mice aged 5 weeks or older were anesthetized, and each material of Example 3-7 was administered via the tail vein. T1 MR images were then acquired at different time points, and SNR was analyzed. As shown in FIG. 9, all the iron-containing nanostructures of Example 3-7 were confirmed to produce a bright signal in the blood vessels (jugular veins) approximately 3-5 minutes after injection.

[412] 3-12. Renal excretion of the iron-containing nanostructures

[413] After intravenous administration of the materials of Example 3-7 to animals, T1-weighted images were acquired by 9.4 Tesla MRI for up to 1 hour to confirm renal excretion of the administered materials. As shown in FIG. 10, a T1 signal began to be observed in the bladder within 30 minutes for most of the iron-containing nanostructures. This indicates that all the administered materials passed through the kidneys and were excreted into the bladder.

[414]

[415] Example 4. MR arthrography using NEMO-103, a crosslinked dextran-based T1 MRI contrast agent

[416] 4-1. Preparation of NEMO-103, a crosslinked dextran-based T1 MRI contrast agent

[417] Dextran T-10 (20 mM) was dissolved in an aqueous solution, and sodium hydroxide and epichlorohydrin (ECH) were sequentially added, followed by ethylenediamine (EDA), to induce crosslinking of the dextran molecules (10,000 g / mol). EDA molecules that did not participate in crosslinking provided terminal amino groups on the core surface, and these amino groups were replaced with carboxyl groups by reaction with succinic anhydride.

[418] The crosslinked dextran thus prepared was purified using a 10 kDa molecular weight cutoff (MWCO) filter, and the purified sample was reacted with aqueous solutions of iron(II) chloride and iron(III) chloride to deposit iron ions onto the carboxyl groups on the core surface. Further purification using a 10 kDa MWCO filter yielded NEMO-103 Injection, a watersoluble, crosslinked dextran-based T1 contrast agent.

[419] NEMO-103 is a colloidal nanoparticle with an amorphous structure in which iron (Fe) is bound to a crosslinked dextran core. The mean hydrodynamic diameter of the prepared particles was 4.0 ± 0.1 nm, and the surface charge was measured to be -11.63 ± 2.7 mV (based on DLS analysis). Approximately 60% or more of the monosaccharide were modified with a crosslinking agent at their -OH functional groups, and this high degree of crosslinking prevented particle aggregation and imparted structural stability through the formation of a high-density three-dimensional network.

[420] This contrast agent remained stable without aggregation for 11 days or longer not only under normal physiological conditions but also at various pH values (5, 7, and 9) and salt concentrations (250, 500, and 1,000 mM NaCl), thereby meeting a stability criterion extending well beyond the excretion period of 24-48 hours confirmed in nonclinical studies.

[421] Analysis of magnetic properties using an MPMS (Magnetic Property Measurement System) showed a magnetization value of 6.53 emu / gFe in a 3 T magnetic field. Measurements of T1 and T2 relaxivities as a function of iron concentration in MRI phantom experiments yielded an r2 / ri ratio of 1.3, indicating ideal relaxivity characteristics for a T1 contrast agent.

[422] 4-2. Nonclinical evaluation of the contrast enhancement performance and immunological safety of NEMO-103, a crosslinked dextran-based T1 contrast agent

[423] The contrast agent NEMO-103 (also designated INV-002) is a T1 MRI contrast agent based on water-soluble crosslinked polysaccharide colloidal particles having a compact, spherical, three-dimensional network structure formed by selectively modifying the monosaccharide -OH functional groups of a dextran polymer using an epoxide-based first crosslinking agent, followed by reaction with a second crosslinking agent (e.g., comprising multiple amino groups) within or between molecules.

[424] This structure was designed to provide physicochemical stability and to maximize immune evasion by inhibiting nonspecific binding to recognition receptors (BCRs, PRRs, etc.) on immune cell surfaces. This example presents nonclinical data encompassing both contrast enhancement performance and safety assessments relating to the potential to induce immune responses.

[425] 4-2-1. Nonclinical evaluation of contrast enhancement performance (CNR-based efficacy analysis)

[426] The image enhancement effect of NEMO-103 was evaluated at the following sites based on the contrast-to-noise ratio (CNR):

[427] Evaluation sites: meniscus, anterior cruciate ligament (cruciate), cartilage, and bone.

[428] Mean CNR improvement: an improvement of 120% or more compared with conventional Gd-based contrast agents.

[429] Duration of contrast enhancement: an increase of at least fourfold.

[430] These results suggest that NEMO-103 enables the acquisition of images with high tissue contrast and provides a basis for improving image resolution and the accuracy of joint diagnosis.

[431] 4-2-2. Toxicity evaluation based on immunological safety (GLP toxicity studies and immunological interpretation)

[432] NEMO-103 met the preclinical safety requirements under U.S. FDA guidelines, and the following characteristics indirectly suggest that it is a non-immunogenic particle:

[433] HEK-293 cell cytotoxicity assay: IC50 not determined; no cytotoxicity.

[434] hERG assay: no impairment of cardiac repolarization.

[435] Platelet / plasma mixing test: no coagulation reaction.

[436] Plasma protein binding: a low level was measured, indicating a low potential for tissue accumulation.

[437] In particular, the crosslinked structure of NEMO-103 was designed based on selective modification of monosaccharide -OH functional groups and precisely controlled crosslinking conditions, indirectly suggesting that it is a non-immunogenic particle at the molecular level, as follows:

[438] Avoidance of binding to PRRs and BCRs: reduced binding affinity for receptors that recognize polysaccharide structural specificity.

[439] Inhibition of clustering: no induction of immune signaling because it does not act as a multivalent antigen.

[440] No induction of immune cell activation: no B-cell activation or antibody production even upon repeated administration.

[441] Stability under pathological conditions: no hypersensitivity reactions even in inflammatory lesions.

[442] 4-2-3. Summary of key numerical results from toxicity studies (including exposure multiples relative to the planned clinical dose)

[443] [Table 4] Item Conditions Toxicity reference Exposure      multiple value relative to the clinical dose Acute toxicity Mouse (single intravenous administration) MTD 42.5 mg / kg 1214-fold Acute toxicity Beagle (single intravenous administration) MTD 12.0 mg / kg 342-fold Repeated-dose toxicity Beagle (intravenous, 28 days) NOAEL       1.6 mg / animal / day 228-fold Repeated-dose toxicity Rat (intravenous, 14 days) NOAEL      6.0 mg / kg / day 857-fold Reproductive toxicity Rat          (intravenous, DRF / perinatal) NOAEL     0.34 mg / animal / day 457-fold

[444] In addition, the reverse mutation test, chromosomal aberration test, and micronucleus test all yielded negative results, and it was concluded that there were no concerns regarding carcinogenicity, genotoxicity, or reproductive toxicity.

[445] Example 4-2 indirectly demonstrated that NEMO-103, a T1 contrast agent based on non-immunogenic crosslinked polysaccharide colloidal particles, is a next-generation MRI contrast agent that provides improved contrast enhancement performance compared with conventional Gd contrast agents while ensuring immunological safety. In particular, the absence of immune responses even upon repeated administration and the absence of observed toxic reactions even at high doses suggest that the platform technology of the present invention is broadly applicable to various bioimaging and precision diagnostic fields.

[446] 4-3. Comparison of image quality in shoulder MR arthrography

[447] To evaluate the clinical efficacy of NEMO-103, image quality following administration of NEMO-103 into the shoulder joint cavity was compared in 32 patients in the Phase 1 / 2a trial (a total of 80 MRA images).

[448] Comparison 1: Comparison of image quality between Phase I (30 minutes) and Phase II (60 minutes) images from the same patients showed no significant differences in CNR, joint capsule distension, or overall image quality (FIG. 11).

[449] Comparison 2: In the comparison of NEMO-103-based and GBCA-based images at the Phase I time point, CNR was similar, but distension of the inferior joint capsule and axillary pouch was significantly greater with NEMO-103, and the overall image quality assessment 79 also showed statistically superior results (p < 0.05, FIG. 12).

[450] Comparison 3: The differences were more pronounced at the Phase II time point (approximately 54-55 minutes), with NEMO-103-based images being significantly superior to GBCA-based images in CNR, inferior joint capsule distension, and axillary pouch distension (p < 0.01, FIG. 13).

[451] 4-4. Visual Turing Test (VTT)

[452] Radiologists were evaluated for their ability to distinguish between images acquired at two different time points (Phase I and Phase II) after injection of NEMO-103. The mean discrimination accuracy among a total of eight readers was 46.8% (146 / 312), which was not statistically significantly different from random guessing (p = 0.423).

[453] 4-5. Safety evaluation in the Phase 1 / 2a clinical trial

[454] The safety of NEMO-103 was evaluated in a clinical trial involving 32 patients with shoulder pain and suspected rotator cuff lesions (Stage 1: n = 9; Stage 2: n = 23).

[455] 4-5-1. Adverse reactions:

[456] Two adverse reactions (ADRs) occurred, both of which resolved, and there were no serious adverse events (SAEs) or drug-related withdrawals. No dose-limiting toxicity (DLT) was observed.

[457] 4-5-2. Retention and tissue safety:

[458] Tissue retention of NEMO-103 in both the liver and spleen was confirmed to be negative (100% disappearance) 24 hours after administration, which was also confirmed by imaging. The differences in liver and spleen MRI signals before and after administration were positive (liver: 13.2 ± 4.0; spleen: 5.5 ± 0.9).

[459] 4-5-3. Image quality and persistence:

[460] Similar image quality was achieved in images acquired at both 30 minutes and 60 minutes after administration, with excellent performance in the depiction of anatomical structures on imaging (joint capsule, rotator cuff, cartilage, etc.). In particular, NEMO-103, which has a long residence time, maintained excellent CNR even after 2 hours and was completely eliminated from the joint cavity after 24 hours, ruling out the possibility of retention in the body.

[461] 4-6. Evaluation of the correlation between joint distension and MRA image quality in the Phase 2b clinical trial

[462] This example is based on the results of a Phase 2b clinical trial conducted to evaluate the intra-articular distribution and contrast enhancement effects of NEMO-103, a T1 MRI contrast agent based on dextran crosslinking. Joint distension, sharpness of joint boundaries, and image contrast were established as the principal image quality assessment parameters in this trial, and statistical significance (p-value < 0.0001) was confirmed in both the primary and supplementary analysis populations (see FIG. 14).

[463] As described below, this example demonstrated the efficacy and stability of NEMO-103 as a T1 contrast agent that provides high-resolution intra-articular MRA images in clinical use and, in particular, showed the potential for AI-based quantitative image analysis based on the correlation between joint distension and image quality.

[464] 1. Intra-articular contrast agent distribution and diagnostic value

[465] Joint distension is an imaging metric that quantifies the uniformity of distribution and extent of penetration of a T1 contrast agent, enhances visualization of intra-articular anatomical structures (cartilage, synovium, ligaments, etc.), and increases diagnostic sensitivity and specificity. In this trial, improved joint distension was closely associated with improvements in image sharpness and contrast and indirectly reflects the uniformity of contrast agent distribution in vivo and prolonged residence time.

[466] 2. Clinical evidence of immune inactivity

[467] NEMO-103 was designed as a contrast agent based on non-immunogenic crosslinked polysaccharide colloidal particles, and this characteristic was also supported in this trial by the following indicators of immunological safety:

[468] Absence of acute immune responses: No abnormal findings attributable to immune responses, such as synovitis, edema, or pain, were observed after administration into the joint cavity.

[469] Maintenance of normal joint distension: The stable increase in distension without excessive distension or reactions within the joint cavity indirectly suggests the absence of immune hypersensitivity.

[470] Uniformity of contrast agent distribution and imaging stability: Together with the physicochemical properties of the contrast agent, stable image quality can be achieved even in an inflammatory environment in vivo.

[471] 3. Potential for AI-based image analysis and standardization

[472] Quantitative imaging metrics, including joint distension, sharpness, and contrast, may be used as a standard dataset for the future development of AI-based image interpretation and automated diagnostic tools. In particular, the quantitative metrics obtained in this trial enhance the reliability of contrast agent evaluation by serving as criteria for reproducible image quality.

[473]

[474] Example 5. Magnetic resonance lymphangiography using INV-001

[475] 5-1. Preparation and characterization of contrast agents

[476] Gd-DOTA (control group):

[477] DOTAREM® (Guerbet, France) was diluted with physiological saline before use (final concentration: 15 mM).

[478] INV-001 (test group):

[479] Dextran T-5 (20 mM) having an average molecular weight of 5,000 Da was reacted with sodium hydroxide and epichlorohydrin, followed by the addition of diethylenetriamine to prepare crosslinked dextran. The terminal amino groups were then modified into carboxyl groups using succinic anhydride, and iron was coordinated to the surface by reaction with iron(III) chloride. The product was purified and concentrated using a 3 kDa MWCO filter.

[480] Structural characteristics:

[481] TEM analysis showed that INV-001 comprised spherical particles having a mean diameter of 2.6 ± 0.3 nm (n = 100), and negative staining was performed using uranyl acetate. Dynamic light scattering analysis showed a hydrodynamic size of 3.6 ± 0.2 nm, and the surface charge was measured to be -2.95 ± 0.20 mV. Approximately 60% or more of the total number of monosaccharides constituting INV-001 were modified by a crosslinking agent, and an average of five iron atoms per particle was confirmed.

[482] Physicochemical characteristics:

[483] Magnetization: 14.25 emu / gFe (3 Tesla magnetic field).

[484] Relaxivity: ri = 3.95 ± 0.35 mM^s"', n = 4.73 ± 0.24 mM-'s1, n / ri = 1.20 ± 0.04.

[485] Other properties: pH = 8.23, viscosity = 3.23 ± 0.03 cP, density = 1.031 ± 0.001 g / cm3, boiling point = 101°C, freezing point = 0°C.

[486] Stability evaluation:

[487] INV-001 maintained a stable colloidal state without aggregation even at pH 5, 7, and 9 and at NaCl concentrations of 250-1,000 mM, and no change in particle size occurred during the period from administration into the body to excretion.

[488] 5-2. Hydrodynamic size and magnetic resonance relaxivity

[489] The hydrodynamic size of INV-001 was measured to be 3.6 nm, and Ti and T2 relaxation rates were measured at various concentrations (0.125, 0.25, 0.5, and 1.0 mM) under a 9.4 T magnetic field. The results were ri = 2.61 mM-1s-1, r2 = 4.33 mM-1s-1, and r2 / ri = 1.66.

[490] This means that INV-001 is suitable as a contrast agent that is specifically retained in the lymphatic system without venous contamination because its size exceeds the maximum size permitting entry into veins (2 nm).

[491] 5-3. Comparison of imaging performance: INV-001 vs. Gd-DOTA (see FIG. 15)

[492] In this example, magnetic resonance lymphangiography (MRL) was performed in Sprague-Dawley (SD) rats to compare the pharmacokinetic characteristics within the lymphatic system and imaging performance of the iron-based contrast agent INV-001 and the gadolinium-based contrast agent Gd-DOTA. In particular, INV-001 is a novel contrast agent designed to prevent venous contamination, and this study evaluated the optimal dose and image quality for visualization of lymph nodes and lymphatic vessels.

[493] After administration of INV-001 and Gd-DOTA at 1.125 pmol (75 pL, 15 mM) each, the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of lymph nodes and lymphatic vessels were compared.

[494] [Table 5] Contrast agent Time SNR (LN) SNR (LV) CNR (LN) CNR (LV) Gd-DOTA 16 min 26.96 ± 10.78 11.15 ± 5.83 21.61 ± 10.04 7.89 ± 5.15 INV-001 16-32 min 23.49 ± 8.64 16.05 ± 3.52 15.87 ± 11.05 12.58 ± 3.37

[495] Whereas Gd-DOTA showed a peak signal at 16 minutes after administration, INV-001 maintained a uniform high signal over a longer period (16-32 minutes), showing superior contrast enhancement particularly in lymphatic vessels (CNRLV).

[496] These results confirmed the promise of INV-001 as an iron-based MRL contrast agent capable of providing less venous contamination than Gd-based contrast agents and stable, prolonged contrast enhancement in lymph nodes and lymphatic vessels.

[497] 5-4. Qualitative comparison of venous contamination, dermal backflow, and lymph node congestion

[498] In this example, INV-001 or Gd-DOTA was injected subcutaneously into the hindlimbs of Sprague-Dawley rats, followed by 3D TOF-based magnetic resonance lymphangiography (MRL) to qualitatively assess the presence or absence of venous contamination, dermal backflow, and lymph node congestion. The assessment was performed according to the qualitative assessment criteria in Table 6.

[499] [Table 6] Description Qualitative score: 0 Qualitative score: 1 Qualitative score: 2 Qualitative score: 3 Venous contamination Present Absent - - Dermal backflow Present Absent - - Lymph      node congestion Present Absent - - Visualization Poor Moderate Good Excellent

[500] Venous contamination was observed in both the 1.125 ^mol and 2.25 ^mol Gd-DOTA groups and was most pronounced in the 2.25 pmol group. In contrast, INV-001 provided good visualization of lymph nodes and lymphatic vessels without venous contamination under all administration conditions. In addition, tissue examination using methylene blue 2 days after MRL imaging confirmed that the lymphatic structures observed in the INV-001-based images 84 corresponded to the actual anatomical structures.

[501] 5-5. Qualitative evaluation of image quality under various injection conditions

[502] To comprehensively evaluate venous contamination and image quality, INV-001 and Gd-DOTA were injected subcutaneously under various conditions, followed by acquisition of MRL images of the popliteal lymph nodes and lymphatic vessels and qualitative scoring according to Table 6.

[503] When the concentration was varied at a fixed injection volume of 75 pL, the INV-001 groups receiving 0.75 pmol (10 mM), 1.125 pmol (15 mM), and 1.5 pmol (20 mM) achieved excellent scores of 4.2, 5.0, and 5.0, respectively. In contrast, the group receiving 2.25 pmol (30 mM) Gd-DOTA showed a lower score of 3.8.

[504] When the injection volume was varied at a fixed concentration of 15 mM, the INV-001 groups receiving 0.3 pmol (20 pL), 0.45 pmol (30 pL), and 0.75 pmol (50 pL) showed scores of 3.6, 4.4, and 4.6, respectively. Gd-DOTA at 1.125 pmol (75 pL) achieved a score of only 3.2.

[505] As shown in FIG. 16, INV-001-based MRL images showed no venous contamination or dermal backflow and provided superior image contrast in lymph nodes and lymphatic vessels compared with Gd-DOTA. In particular, the highest level of image quality was confirmed at doses of 0.45 pmol and 0.75 pmol.

[506] Example 5-5 experimentally demonstrated that the iron-based contrast agent INV-001 has superior biocompatibility and lymphatic vessel-specific imaging characteristics compared with Gd-DOTA. In particular, INV-001 has a hydrodynamic diameter of approximately 3.6 nm, exceeding the size limit for entry into veins (approximately 2 nm), thereby fundamentally preventing venous contamination and provided prolonged visualization of lymph nodes and excellent contrast enhancement. Biological safety evaluation showed no residual INV-001 in tissues within 24 hours and 48 hours after administration and no changes in liver or kidney signals, confirming rapid elimination from the body.

[507] Accordingly, INV-001 has strong potential for use as a T1 contrast agent for magnetic resonance lymphangiography (MRL) and is a promising candidate capable of providing stable image quality over an extended period without venous contamination in the diagnosis and monitoring of lymphatic diseases.

[508] 5-6. Evaluation of the lymphatic vessel contrast enhancement efficacy of INV-001 in various animal models

[509] In this example, the lymphatic vessel-specific contrast enhancement capability of INV-001 was evaluated in various animal models (Sprague-Dawley rats, minipigs, and beagle dogs) and compared with that of a gadolinium-based contrast agent (Gd-DOTA). Each experiment was evaluated primarily in terms of the degree of lymphatic vessel visualization and the presence or absence of venous contamination, and the image analysis results are shown in FIG. 17.

[510] 1) Sprague-Dawley rats (MRL imaging)

[511] INV-001 or Gd-DOTA was injected subcutaneously into the hindlimbs of SD rats, followed by 3D TOF-based MRL imaging. In the INV-001-injected group, even peripheral lymphatic vessels with diameters of 300 pm or less were clearly visualized, and no venous contamination was observed. In contrast, venous contamination was clearly identified when Gd-DOTA was administered under the same conditions.

[512] 2) Minipig model

[513] MRL was performed after injection of INV-001 or Gd-DOTA under conditions corresponding to the low dose in the clinical trial (15 mM, 0.6 mL x 3 sites). Following administration of INV-001, abdominal lymph nodes and lymphatic vessels in the legs were clearly visualized, and no venous contamination was observed. In contrast, following administration of Gd-DOTA, lymphatic vessel branches appeared faint, and venous structures were contrast-enhanced, resulting in clearly observed contamination.

[514] 3) Beagle dog model

[515] Visualization of lymphatic vessels after injection of INV-001 into the dorsum of the foot showed clearly delineated lymphatic vessels. In particular, compared with the untreated control group, lymphatic vessel structures were observed only in the INV-001-treated group. This suggests that the contrast enhancement effect of INV-001 effectively extended to cutaneous and intramuscular lymphatic vessels.

[516] 4) Verification of efficacy in a disease model

[517] In a minipig disease model involving lymph node resection, lymphatic vessel contrast enhancement was compared between the left-side lymph node resection group and the rightside normal group after injection of INV-001. The images showed no identifiable lymph nodes on the resected left side, whereas lymphatic vessel branches and lymph nodes were clearly contrast-enhanced in the right-side normal group. This shows that INV-001 can sensitively reflect changes in lymphatic structures even under disease conditions.

[518] 5-7. Observation of contrast enhancement extending to the central lymphatic system after peripheral administration of INV-001 in beagle dogs

[519] In this example, the iron-based T1 MRI contrast agent INV-001 was injected subcutaneously into the distal hindlimbs of beagle dogs, and magnetic resonance lymphangiography (MRL) was used to evaluate whether the contrast agent reached not only the peripheral lymphatic system but also the central lymphatic system.

[520] As shown in FIG. 18, INV-001 ascended from the injection site in the distal hindlimb along the popliteal lymph nodes and peripheral lymphatic vessels and produced extensive contrast enhancement extending to the central lymphatic vessel region in the abdomen. In particular, spread to the central lymphatic system was observed without signal loss in the lymph nodes, suggesting that the contrast agent can continue to move with lymph flow without being captured by immune cells in the lymph nodes.

[521] These results indirectly demonstrate that interactions of the contrast agent components of INV-001 with immune cells in the body, particularly immune cells in lymph nodes such as B cells and dendritic cells, are markedly inhibited. This is attributable to the structural characteristics of the non-immunogenic crosslinked polysaccharide colloidal particles of the present invention. Specifically, binding affinity for PRRs or BCRs on immune cells is reduced by (a-1) selectively modifying monosaccharide -OH functional groups with an epoxide-based first crosslinking agent, (a-2) forming a three-dimensional network between the modified -OH functional groups using a second crosslinking agent (e.g., a polyamine) or through a direct reaction, and (a-3) further modifying crosslinker-derived amino groups into carboxylic acid groups to adjust the surface charge to a range of -20 mV to 0 mV.

[522] As a result, INV-001 (i) minimizes receptor-ligand interactions with numerous immune cells in lymph nodes, such that signaling through receptor clustering is not induced, and (ii) does not induce B-cell activation or antibody production. While (iii) maintaining resistance to hydrolysis by enzymes in the body, it (iv) can undergo stable elimination without 87 hypersensitivity immune responses even under inflammatory conditions.

[523] These technical characteristics, as demonstrated by the imaging experiment in FIG. 18, show that INV-001 can traverse lymph node structures densely populated with immune cells and reach the central lymphatic system along the flow of lymph, strongly supporting the applicability of the contrast agent of the present invention as a non-immunogenic T1 lymphatic contrast agent platform.

[524] 5-8. GLP nonclinical toxicity evaluation and verification of the immune evasion design of INV-001, a non-immunogenic crosslinked polysaccharide colloidal contrast agent

[525] This example summarizes the results of nonclinical toxicity studies conducted in accordance with GLP standards to confirm that INV-001, the iron-based T1 contrast agent of the present invention, exhibits excellent biological safety under high-dose and repeated-administration conditions without inducing immune responses in the body.

[526] Conventional polysaccharide-based drugs or nanoparticles may interact with immune cells (B-cell receptors, pattern recognition receptors, etc.) and induce unexpected immune responses (e.g., antibody production, inflammation, hypersensitivity reactions, and allergy). In particular, contrast agents based on crosslinked polysaccharides may present problems of accumulation in the body and long-term retention attributable to their polymeric structures, raising concerns about immunotoxicity and reduced contrast agent efficacy.

[527] INV-001 of the present invention was designed as a non-immunogenic crosslinked polysaccharide colloidal particle platform to avoid B-cell activation and antibody production, inhibit receptor clustering, and allow stable elimination without retention in organs.

[528] As shown in the table below, no toxic reactions to INV-001 were observed in the cardiovascular, central nervous, respiratory, or immune systems or in major organ systems, even at high doses several tens of times the planned clinical dose, and complete elimination without accumulation in the body was confirmed.

[529] [Table 7] Item Test conditions Exposure       multiple relative to the clinical dose Central nervous system / respiratory system GLP toxicity study 268-fold Cardiovascular system (telemetry) Beagle dogs 116-fold Cardiovascular system (hERG) in vitro Acceptable Pharmacokinetics (absorption / distribution / excretion) Included in toxicity studies 35-fold MTD (Mouse, intravenous) 49 mg / kg 438-fold MTD (Rat, subcutaneous) 0.52 mg / animal 23-fold MTD (Beagle, subcutaneous) 6.24 mg / kg 7-fold NOAEL (Beagle, repeated administration for 28 days, subcutaneous) 6.24 mg / kg 7-fold

[530] In addition, negative results in the reverse mutation test, chromosomal aberration test, and micronucleus test also ruled out the possibility of genotoxicity.

[531] INV-001 of the present invention employs a molecular design strategy that blocks nonspecific binding to immune cell receptors to address the immunological and pharmacokinetic limitations of conventional gadolinium-based contrast agents. The immune evasion and reduced retention properties of this contrast agent were demonstrated by imaging results showing its stable transport to the central lymphatic system even after passage through lymph nodes, without being phagocytosed by immune cells or becoming retained (see FIG. 18 and Example 5-7).

[532] Furthermore, the GLP study results demonstrated that it is a non-immunogenic contrast agent that is safely excreted without antibody production or tissue reactions even upon repeated administration and that markedly reduces the potential for immune-mediated adverse effects (allergy, inflammation, etc.) associated with polysaccharide-based drugs.

[533] This example confirmed that INV-001 is a contrast agent with a high degree of safety that is excreted without accumulating in vivo and that suppresses immune activation upon repeated administration based on structural characteristics that reduce binding affinity for receptors on immune cells. Accordingly, INV-001 can be used as a lymphatic-specific T1 contrast agent with a low risk of inducing immune responses and with safety ensured even under high-dose and repeated-administration conditions.

[534]

[535] Example 6. Evaluation of lymphatic system-specific imaging efficacy and safety based on immune inactivity in the Phase 1 clinical trial of INV-001

[536] This example concerns the results of a Phase 1 clinical trial conducted to evaluate image quality and safety, particularly immune system responsiveness, when INV-001 is used in humans.

[537] High-resolution magnetic resonance lymphangiography (MRL) was performed after intradermal or subcutaneous administration of INV-001 to the distal region of the foot. INV-001 moved from the injection site along peripheral lymphatic vessels, and clear, sharp lymphatic vessel images were observed (see FIG. 19). The images reflected the distribution of the contrast agent within the lymphatic system and enabled lymphatic system-specific, high-resolution visualization over time after injection.

[538] In parallel, the safety of INV-001 was evaluated in a Phase 1 clinical trial involving three dose groups (0.7 mL / site, 1.0 mL / site, and 1.3 mL / site), with administration to a total of nine healthy adult volunteers (see Table 8).

[539] [Table 8] Dose Age / Sex Dose Level (mL / site) AE SAE DLT NRS pain scale Low dose 68 / M 0.7 None None None 1 / 1 / 1 37 / F 0.7 None None None 3 / 1 / 1 53 / F 0.7 None None None 6 / 1 / 1 Medium dose 33 / F 1.0 None None None 9 / 1 / 0 25 / F 1.0 None None None 4 / 0 / 0 49 / F 1.0 None None None 8 / 1 / 0 High dose 53 / F 1.3 None None None 0 / 0 / 0 27 / F 1.3 None None None 0 / 0 / 0 24 / F 1.3 None None None 0 / 0 / 0

[540] The following key indicators relating to immunological safety were confirmed:

[541] Maximum tolerated dose (MTD) not reached: A toxicity-defined maximum tolerated dose (MTD) was not observed in any dose group, suggesting that immune system-related adverse reactions, such as systemic inflammatory responses, febrile reactions, and cytokine storms, did not occur to a significant extent even at high doses.

[542] No adverse drug reactions (ADRs) or serious adverse events (SAEs): No drug-related adverse reactions (ADRs) or serious adverse events (SAEs) were observed in any study participant. This means that immune-mediated adverse effects, such as allergy, rash, and vasculitis, were not clinically identified.

[543] Successful lymphatic system-specific imaging: Although lymph nodes and lymphatic vessels are tissues densely populated with immune cells, INV-001 provided stable contrast enhancement of lymph nodes, and no image degradation or local inflammatory responses due to interactions with immune cells were observed. This indirectly shows that INV-001 was not recognized as an immunogenic substance by T cells, B cells, macrophages, or other cells in lymph nodes.

[544] In particular, INV-001 is a water-soluble crosslinked polysaccharide colloidal particle having a compact, spherical, three-dimensional network structure formed by selectively modifying monosaccharide -OH functional groups and then precisely controlling the crosslinking agent conditions. Its binding affinity for immune cell receptors, such as B-cell receptors (BCRs) and PRRs, is markedly low, and its potential to induce immune responses is therefore very low. These structural characteristics are directly linked to the nonimmunogenicity confirmed by the Phase 1 clinical results.

[545] The above results suggest that both the safety and efficacy of INV-001 as a T1 MRI contrast agent capable of precisely visualizing the lymphatic system in humans without immune system-related adverse reactions have been demonstrated, supporting its potential for future clinical use in the diagnosis and monitoring of lymphatic diseases.

[546]

[547] Example 7. INV-MRL-based contrast enhancement technology for lymphedema staging: application of non-immunogenic crosslinked polysaccharide colloidal particles

[548] This example describes the results of effective visualization of lymphatic lesions and application to disease staging in an animal model of lymphedema using magnetic resonance lymphangiography (hereinafter, INV-MRL) with INV-001, a contrast agent based on chemically crosslinked, non-immunogenic crosslinked polysaccharide colloidal particles.

[549] The contrast agent used in this example comprises colloidal particles having a highly precise three-dimensional network structure formed by selectively modifying monosaccharide -OH functional groups using an epoxide-based first crosslinking agent and then inducing crosslinking between adjacent -OH functional groups using a second crosslinking agent (comprising a polyamine) or through a direct crosslinking reaction. These particles were designed to avoid interactions with immune cells in vivo (e.g., BCRs, PRRs, etc.) by replacing some or all of the crosslinker-derived amino groups with -COOH functional groups to adjust the surface charge to within a range of -20 mV to 0 mV. This structural design prevents clustering of immune cell receptors and confers non-immunogenic properties that do not induce immune activation even upon repeated administration.

[550] FIG. 20 shows representative images obtained by applying INV-MRL and NIRF-ICGL, respectively, in an animal model of induced lymphedema. INV-MRL clearly visualizes deep lymphatic vessels even in regions where the contrast agent spreads along the skin surface, suggesting an advantage in detecting structural abnormalities that are difficult to identify using NIRF-ICGL. In particular, as indicated by the orange arrows in FIG. 20, INV-MRL was able to image even obscured collateral lymphatic vessels (collateral LVs) beneath the spread of contrast agent.

[551] INV-MRL also enabled qualitative identification of structural abnormalities, such as lymph leakage, formation of collateral pathways, and lymphatic vessel dilation, according to lymphedema stages classified from 0 to 4. These abnormalities showed a high correlation with the splash, stardust, and diffuse patterns defined in NIRF-ICGL. TAR (Thresholded Area Ratio) values derived from the same ROI showed a quantitative correlation, increasing with disease stage progression, and TAR values measured by INV-MRL showed higher sensitivity than those measured by NIRF-ICGL (R2 = 0.5919 vs. 0.5309, dorsal NIRF).

[552] In particular, INV-MRL provides the following technical advantages:

[553] Retention in immune organs, such as lymph nodes, is limited because it is based on non-immunogenic crosslinked polysaccharide colloidal particles.

[554] Unlike GBCA-based MRL, it selectively visualizes only lymph flow without venous contamination.

[555] It has low dependence on ROI and enables reproducible visualization even of deep lymphatic structures under the same conditions.

[556] Its 3D reconstruction capability enables quantitative analysis of the extent of lymph spread with disease stage progression.

[557] Through this technical verification, INV-MRL was evaluated as a superior imaging platform capable of visualizing deep lymphatic structures compared with conventional optical imaging-based NIRF-ICGL, demonstrating the applicability of non-immunogenic crosslinked polysaccharide colloidal particles to lymphatic diagnostics.

[558] This example demonstrates at the preclinical level that the design rationale and structural characteristics of non-immunogenic crosslinked polysaccharide particles, which are key technical components of the present invention, can provide a new paradigm for lymphatic imaging technology, and provides a technical basis for establishing new standards for high-precision lymphatic imaging and disease staging in future clinical applications.

Claims

1. A non-immunogenic, water-soluble crosslinked polysaccharide colloidal particle in which -OH functional groups of a polysaccharide to be cross-linked, dispersed in an aqueous solvent, are modified with a crosslinking agent and the surface charge of the particle is adjusted to within a range of -20 mV to 0 mV through surface-exposed -COOH functional groups, thereby reducing or minimizing immunogenicity toward immune cells compared with the polysaccharide to be cross-linked and allowing the particle to be eliminated from the body without being hydrolyzed by enzymes in the body, wherein the crosslinked polysaccharide colloidal particle is formed in an aqueous solvent by:(i) modifying -OH functional groups of monosaccharides constituting the building blocks of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide with a first crosslinking agent having an epoxide group, and forming a crosslinked polysaccharide particle through intramolecular and / or intermolecular crosslinking (a) directly between a functional group modified with the first crosslinking agent and a spatially adjacent -OH functional group and / or (b) between two spatially adjacent functional groups modified with the first crosslinking agent via a second crosslinking agent having two or more amino groups (-NH2); and(ii) adjusting the number of surface-exposed crosslinker-derived basic amino groups through modification into -COOH functional groups.

2. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 1, wherein 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides constituting the building blocks of the polysaccharide are modified with a crosslinking agent such that the particle is not hydrolyzed by enzymes in the body and its immunogenicity toward immune cells is reduced or minimized.

3. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 1, wherein at least one -OH functional group in at least one of two consecutive monosaccharides within a polysaccharide chain is predominantly modified with a crosslinking agent, such that the crosslinked polysaccharide colloidal particle is not hydrolyzed by enzymes in the body and its immunogenicity toward immune cells is reduced or minimized.

4. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 1, wherein the crosslinked polysaccharide colloidal particle (a) is filtered by the kidneys butcannot pass through the vascular walls of normal capillaries and optionally (b) is drained exclusively into peripheral lymphatic vessels, and has a hydrodynamic diameter of 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less.

5. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 4, wherein, after administration into the body, the crosslinked polysaccharide colloidal particle enters the blood vessels of the nephron and is excreted in urine through the renal filtration mechanism.

6. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 4, wherein the crosslinked polysaccharide colloidal particle is selectively drained exclusively into peripheral lymphatic vessels without entering or being drained into capillaries at the administration site, and an amount corresponding to 50% or more of the injected amount remains at the administration site for 1 hour or longer, preferably 2 hours or longer, before drainage through the lymphatic vessels.

7. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 6, wherein a prolonged residence time at the injection site induces an increase in interstitial space (interstitial space enhancement) and / or anatomical space distension at the injection site.

8. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 1, wherein the crosslinked polysaccharide colloidal particle itself is not taken up by cells unless targeted with a ligand having affinity for a cell surface receptor.

9. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 1, wherein iron ions, gadolinium ions (Gd3+), manganese ions (Mn2+), or iron oxide particles are coordinately bonded to surface-exposed crosslinker-derived amino groups or -COOH functional groups of the crosslinked polysaccharide colloidal particle, and the particle is used as an MRI contrast agent.

10. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 1, wherein the crosslinked polysaccharide colloidal particle to which metal ions are coordinately bonded simultaneously acts as a chelator of metal ions through crosslinker-derived amino groups or -COOH functional groups to which metal ions are not coordinately bonded.

11. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the particle is provided by a preparation method comprising:a first step of preparing an aqueous solution of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide;a second step of adding dropwise an alkaline aqueous solution and a first crosslinking agent having an epoxide functional group that reacts with hydroxyl groups (-OH) of the polysaccharide, thereby modifying -OH functional groups of monosaccharides constituting the building blocks of the polysaccharide with the first crosslinking agent;a third step of adding dropwise a second crosslinking agent having two or more amino groups (-NH2), thereby causing spatially adjacent functional groups modified with the first crosslinking agent to undergo intramolecular and / or intermolecular crosslinking via the second crosslinking agent and producing crosslinked polysaccharide colloidal particles having terminal amino groups derived from the second crosslinking agent on their surfaces;a fourth step of adding an organic acid anhydride to the crosslinked polysaccharide colloidal particles having terminal amino groups derived from the second crosslinking agent on their surfaces, thereby modifying some or all of the terminal amino groups into carboxylic acid groups and / or carboxylate groups; andoptionally, a fifth step of adding a precursor providing iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+), or an aqueous solution of iron oxide nanoparticles, to the water-dispersible crosslinked polysaccharide colloidal particles prepared in the preceding step, thereby preparing (i) crosslinked polysaccharide colloidal particles having iron ions (Fe2+ / 3+), gadolinium ions (Gd3+), or manganese ions (Mn2+) coordinately bonded to surface-exposed crosslinker-derived amino groups or -COOH functional groups, or (ii) a composite in which the surfaces of iron oxide nanoparticles are modified with crosslinked polysaccharide colloidal particles.

12. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the crosslinked polysaccharide colloidal particle does not elicit an immune response as an antigen, does not induce the production of antibodies against itself as an antigen, can be repeatedly administered, does not cause cell damage or tissue damage due to an inflammatory response, or does not induce a chronic inflammatory disease.

13. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein reduced or minimized immunogenicity toward immune cells meansthat the crosslinked polysaccharide colloidal particle is not recognized as a foreign antigen by the immune system upon distribution in the body, antibodies against the crosslinked polysaccharide colloidal particle are not produced, hypersensitivity reactions or immune-mediated adverse effects do not occur, the complement system is not activated, an inflammatory response is not elicited, the particle is not phagocytosed by macrophages upon repeated administration, the particle is not recognized or bound by pattern recognition receptors (PRRs), cytokines are not released, or the particle does not stimulate the NF-kB activation pathway in macrophages.

14. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 12, wherein inflammation or hypersensitivity reactions do not occur even at a dose that is 2, 3, 4, 5, 6, 7, 8, 9, 10, or 102 times the administered dose.

15. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the crosslinked polysaccharide colloidal particle is a nonimmunostimulatory particle that is not an immunogen or antigen that binds directly to cell membrane receptors on immune cells or indirectly through antibodies to immune cells or non-immune cells bearing Fc receptors.

16. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the crosslinked polysaccharide colloidal particle does not activate or induce proliferation of B cells or mast cells upon distribution in the body.

17. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the crosslinked polysaccharide colloidal particle does not induce one or more immune-mediated adverse effects selected from the group consisting of inflammation, edema, allergy, and hypersensitivity reactions upon administration into the body.

18. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the polysaccharide to be cross-linked is a T-cell-independent multivalent antigen having two or more epitopes.

19. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the crosslinked polysaccharide colloidal particle does not act as a multivalent antigen having two or more epitopes unless it undergoes gelation or aggregation at the administration site through adjustment of the administered dose.

20. The non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10, wherein the surface of the crosslinked polysaccharide colloidal particle is covalently or coordinately bonded to nanoparticles, molecules, or metal ions through crosslinker-derived amino groups or -COOH functional groups.

21. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 20, wherein the number of drug bound to the crosslinked polysaccharide colloidal particle (drug-to-particle ratio, DPR) is controlled in accordance with the number of the crosslinker-derived amino groups.

22. The non-immunogenic crosslinked polysaccharide colloidal particle according to claim 20, wherein the crosslinked polysaccharide colloidal particle, modified with a ligand targeting a cell surface receptor through crosslinker-derived amino groups or -COOH functional groups, is retained at a target site for 1 day or longer, preferably 4 days or longer.

23. A pharmaceutical composition wherein the non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10 is used as a drug carrier for early diagnosis of an inflammatory disease or as a lesion-targeted delivery platform.

24. A pharmaceutical composition wherein the non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10 is used as a contrast agent for quantitative metrics in an AI-based image analysis system.

25. A diagnostic composition wherein the non-immunogenic crosslinked polysaccharide colloidal particle according to any one of claims 1 to 10 is used as a contrast agent capable of differentiating inflammatory lesions or determining their stage.

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