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

Functional drug based on polysaccharide cross-linked colloidal particles that can be released into the lymphatic system and have reduced immunogenicity

[0001] The present invention relates to a functional drug based on polysaccharide cross-linked colloidal particles that can be released into lymphatic vessels and have reduced immunogenicity; and various uses thereof.

[0002] Specifically, the present invention is a water-soluble polysaccharide cross-linked colloidal particle in which the -OH functional group of a polysaccharide as a cross-linking target dispersed in an aqueous solvent is modified with a cross-linking agent and the surface charge of the particle is controlled within a range of -20 mV to 0 mV through the -COOH functional group exposed on the surface, thereby reducing or minimizing immunogenicity for immune cells compared to the polysaccharide as a cross-linking target and being discharged from the body without being hydrolyzed by enzymes in the body, wherein the polysaccharide cross-linked colloidal particle is a polysaccharide cross-linked colloidal particle in which (i) the -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-linking agent having an epoxide group, and (a) the functional group modified with the first cross-linking agent and the spatially adjacent -OH functional group are cross-linked intramolecularly and / or intermolecularly through a second cross-linking agent having two or more amine groups (-NH2) in an aqueous solvent. Polysaccharide cross-linked colloidal particles characterized in that (i) the number of basic amine groups derived from a cross-linking agent exposed on the surface is controlled by modification with -COOH functional groups; and various uses thereof.

[0003] Polysaccharides are essential components along with other biomolecules such as proteins and nucleotides and exert many activities in biological systems such as cell-to-cell communication, adhesion, and molecular recognition in the immune system.

[0004] Dextran is a complex, branched polysaccharide derived from bacteria. When introduced into the body, dextran can be recognized as a foreign antigen by the immune system. Dextran (MW 40 kDa) inhibits platelet aggregation and coagulation factors and is used as a blood plasma volume expander. However, it can cause adverse drug reactions such as pain, inflammation, and edema.

[0005] Macrophages express multiple surface receptors that bind polysaccharides to recognize foreign pathogens or damaged cellular debris. These receptors interact with polysaccharide components (e.g., β-glucan, mannose, fucose, N-acetylglucosamine, etc.) to activate signaling pathways that induce immune responses or modulate inflammatory responses. Representative polysaccharide-binding receptors include TLR4, mannose receptor (CD206), Dectin-1, complement receptor 3 (CR3), and scavenger receptor (SR).

[0006] Ligands of Toll-like Receptor 4 (TLR4) include β-glucan (e.g., fungal cell wall), bacterial lipopolysaccharide (LPS), and ginseng polysaccharide. TLR4 recognizes ligands by forming a complex with CD14 or MD-2, and activates MAPK (ERK1 / 2, JNK, p38) and NF-κB through the MyD88-dependent pathway, promoting the secretion of proinflammatory cytokines such as TNF-α and IL-6. It also increases the activity of the AP-1 transcription factor through a synergistic effect with IFN-γ, thereby amplifying NO and cytokine secretion.

[0007] Ligands of mannose receptor (CD206) include mannose, fucose, and N-acetylglucosamine. Ca is released through the C-type lectin domain (CRD4).2+ -It binds to sugars in a sugar-dependent manner and is primarily involved in endocytosis and antigen presentation. While the direct signaling pathways are not yet fully elucidated, the sugar-binding structure has been analyzed in detail.

[0008] Dectin-1 ligands are fungal polysaccharides, such as β-1,3-glucan. They activate Syk kinase and CARD9, inducing the secretion of IL-1β, IL-6, and ROS via the NF-κB pathway. They also promote the secretion of TNF-α and IL-12 through cooperation with TLR2. For example, it has been reported that the polysaccharide of Inonotus obliquus binds to Dectin-1 and induces tumoricidal activation of macrophages.

[0009] The ligands of complement receptor 3 (CR3; CD11b / CD18) include complement-opsonized pathogens and β-glucan. They cooperate with TLR4 or CD14 to activate the MAPK / AP-1 pathway, inhibiting NO secretion and promoting phagocytosis in LPS-activated macrophages.

[0010] The ligands of scavenger receptors (SRs) are sulfated polysaccharides (e.g., dextran sulfate). They balance proinflammatory and anti-inflammatory responses by inducing the NF-κB and PI3K / AKT pathways. Increased SR expression in BCG-activated macrophages enhances polysaccharide binding capacity.

[0011] Polysaccharide-binding receptors on the surface of macrophages do not function as single receptors, but rather fine-tune the immune response through mutual cooperation and cross-signaling. Macrophages utilize a variety of polysaccharide-binding receptors to eliminate pathogens and control inflammation, and the cooperation of these receptors plays a crucial role in precisely fine-tuning the immune response.

[0012] NF-κB is a transcription factor that plays a crucial role in regulating immune responses, inflammation, and cell survival. NF-κB increases key signaling molecules involved in inflammation and immune responses, as well as cell proliferation, apoptosis, and the cell cycle.

[0013] The typical NF-κB activation pathway, which constitutes a signaling system essential for innate immunity, is a pathway in which NF-κB in the cytoplasm moves into the nucleus and is activated through IKKβ-dependent IκB degradation.

[0014] When cells are stimulated from the outside through pathogen-associated molecular patterns (PAMPs), TNF receptors (TNFRs), Toll-like receptors (TLRs), IL-1 receptors (IL-1R), etc., the IKK complex binds to the NF-κB dimer and inhibits its activation. The IκB is degraded in the proteasome through phosphorylation and polyubiquitination, thereby separating the NF-κB dimer from IκB and activating it. The NF-κB dimer with IκB separated passes through the nuclear membrane (usually a p50-p65 dimer) and enters the nucleus, where it binds to DNA and activates gene transcription. It is known that the typical NF-κB activation pathway activates the transcription of genes encoding various chemokines, cytokines, ICAM-1, VCAM-1, endothelial leukocyte adhesion molecule-1 (ELAM), secondary inflammatory mediators, and apoptosis inhibitors.

[0015] Meanwhile, B cells possess 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, antigens can cross-link multiple BCRs on the B cell surface, leading to activation.

[0016] The NF-κB signaling pathway plays an essential role in B cell survival, proliferation, and immune response regulation, and is closely linked to BCR signaling. This pathway is activated through the canonical IKK-dependent pathway and the alternative (non-IKK-dependent) pathway, and dysregulation of these pathways is associated with various pathological conditions, including lymphoma and autoimmune diseases. NF-κB 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, it promotes MYC and cyclin expression, inducing cell division.

[0017] Chemical cross-linking of polysaccharides leads to the formation of a three-dimensional network structure through a chemical reaction. The physical properties of the particles synthesized by chemical cross-linking polysaccharides, such as size, surface charge, and porosity, significantly influence the biodistribution and in vivo retention time of chemically cross-linked polysaccharide-based drugs. Therefore, even with precise control of synthetic conditions (e.g., reaction temperature, reaction time, pH, reactant concentration, etc.), subtle differences in the degree of cross-linking, molecular weight distribution, and functional group arrangement can occur. This structural heterogeneity leads to variations in particle solubility, physical stability, and interactions with biomolecules (e.g., serum proteins, cellular receptors), resulting in differences in in vivo pharmacokinetic behavior (metabolism, excretion, in vivo retention time, etc.) even for identical dextran-based nanoparticles.

[0018] Furthermore, because polysaccharide structures are polymeric and complex, metabolic and excretory pathways vary across species. Therefore, even chemically cross-linked polysaccharide-based drugs may exhibit metabolic stability or elimination rates in animal models that do not match human metabolic mechanisms. This can lead to unexpected drug accumulation or the formation of toxic metabolites.

[0019] Moreover, chemically cross-linked polysaccharide-based drugs can be recognized differently by the immune system in different species. Specifically, the expression patterns of polysaccharide-recognizing receptors, such as lectins and scavenger receptors, vary significantly between rodents, primates, and humans. For example, while the same polysaccharide-cross-linked nanoparticle is rapidly cleared by the reticular system (RES) in mice, it may persist longer in humans, potentially inducing unexpected complement activation or cytokine secretion.

[0020] When a foreign substance is introduced into the body, it is recognized by innate immune receptors (PRRs) and B cell receptors (BCRs), triggering an immune response. Specifically, B cells cluster in specific spaces, determining signal strength and persistence. This leads to abnormal immune stimulation, which promotes B cell proliferation, differentiation, and antibody production, potentially leading to allergies, inflammation, and hypersensitivity reactions. Existing drug delivery systems have limitations in completely addressing the side effects caused by immunogenicity, such as rapid carrier clearance and reduced therapeutic efficacy.

[0021] Conventional polysaccharide-based drugs and nanoparticles often trigger recognition through immune cell receptors, resulting in rapid clearance from the body, side effects, and reduced therapeutic efficacy. In particular, differences in immune receptor expression and metabolic pathways between animal models and humans have raised concerns about unpredictability.

[0022] The present invention seeks to provide non-immunogenic polysaccharide-crosslinked colloidal particles that can solve the problems of unpredictability of biological activity due to the aforementioned in vivo pharmacokinetic behavior (metabolism, excretion, body retention time, etc.) and interaction with biomolecules (e.g., polysaccharide recognition receptors, BCRs) related to chemically crosslinked polysaccharide-based drugs.

[0023] Even if the same polysaccharide is chemically cross-linked with the same degree of cross-linking, the binding to the surface receptor of an immune cell may vary depending on the species. By taking advantage of this problem, the present invention selectively modifies the -OH functional group of a monosaccharide with an epoxide-based first cross-linking agent to chemically cross-link and precisely controls the cross-linking conditions (e.g., type and amount of cross-linking agent, reaction temperature, reaction time, pH, concentration of reactants, etc.) even when various polysaccharide polymers are cross-linked, thereby minimizing the interaction with the surface receptor of an immune cell even with slight differences and suppressing immune activation even upon repeated administration. It is intended to provide a non-immunogenic polysaccharide cross-linked colloidal particle platform having a compact, spherical three-dimensional network structure.

[0024] In addition, the present invention aims to provide a non-immunogenic polysaccharide cross-linked colloidal particle platform technology that can effectively overcome the immune response and resulting adverse reactions (e.g., allergy, inflammation, hypersensitivity, edema, etc.) generally induced by polysaccharide-based drugs or nanoparticles administered into the body, as well as the reduced efficacy of therapeutic agents or contrast agents.

[0025] Furthermore, the present invention seeks to provide polysaccharide cross-linked colloidal particles that have reduced or minimized immunogenicity against immune cells compared to polysaccharides, which are cross-linking targets that can cause immune side effects when administered in the body, and are excreted from the body without being hydrolyzed by enzymes in the body.

[0026] In addition, the present invention seeks to provide polysaccharide cross-linked colloidal particles that (a) are filtered in the capillaries of the kidney but do not penetrate the walls of normal capillaries and (b) are optionally excreted only into the distal lymphatic vessels.

[0027] The first aspect of the present invention is a water-soluble polysaccharide cross-linked colloid particle in which the -OH functional group of the cross-linking target polysaccharide dispersed in an aqueous solvent is modified with a cross-linking agent and the surface charge of the particle is controlled within the range of -20 mV to 0 mV through the -COOH functional group exposed on the surface, thereby reducing or minimizing immunogenicity to immune cells compared to the cross-linking target polysaccharide and being excreted from the body without being hydrolyzed by enzymes in the body.

[0028] The present invention provides a non-immunogenic polysaccharide cross-linked colloidal particle characterized in that (i) the -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-linking agent having an epoxide group in an aqueous solvent, (a) the functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group are cross-linked intramolecularly and / or intermolecularly via a second cross-linking agent having two or more amine groups (-NH2), thereby forming the polysaccharide cross-linked particle, and (ii) the number of basic amine groups derived from the cross-linking agent exposed on the surface is controlled by modification with a -COOH functional group.

[0029] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention may be modified by a cross-linking agent in which at least 60%, at least 70%, at least 90%, or at least 95% of the total number of monosaccharides, which are building blocks of the polysaccharide, are modified by a cross-linking agent so that they are not hydrolyzed by enzymes in the body and immunogenicity against immune cells is reduced or minimized.

[0030] In addition, the non-immunogenic polysaccharide cross-linked colloidal particle of the present invention may be one in which at least one -OH functional group in at least one of two consecutive monosaccharides in a polysaccharide chain is mostly modified with a cross-linking agent, thereby preventing hydrolysis by enzymes in the body and reducing or minimizing immunogenicity to immune cells.

[0031] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention, when having a hydrated diameter of 2 to 10 nm, preferably 8 nm or less, more preferably 6 nm or less, (a) are filtered in the capillaries of the kidney, but do not penetrate the walls of normal capillaries, and (b) can be optionally excreted only into the distal lymphatic vessels.

[0032]

[0033] The second aspect of the present invention provides a pharmaceutical composition characterized by using the non-immunogenic polysaccharide cross-linked colloidal particles of the first aspect as a drug delivery vehicle for early diagnosis of inflammatory diseases or a lesion-targeted delivery platform.

[0034] The third aspect of the present invention provides a pharmaceutical composition characterized in that the non-immunogenic polysaccharide cross-linked colloidal particles of the first aspect are used as a contrast agent for quantitative indicators of an AI-based image analysis system.

[0035] The fourth aspect of the present invention provides a diagnostic composition characterized in that the non-immunogenic polysaccharide cross-linked colloidal particles of the first aspect are used as a contrast agent capable of differentiating or staging inflammatory lesions.

[0036]

[0037] Hereinafter, the present invention will be described.

[0038] Traditionally, when foreign substances are administered into the body, they are recognized by surface receptors on immune cells (e.g., innate immune receptors (PRRs), B-cell receptors (BCRs), etc.), triggering immune responses and adverse effects (allergies, inflammation, hypersensitivity, edema, etc.). These immune responses can lead to rapid clearance of drug delivery vehicles and reduced efficacy of therapeutic agents or contrast agents. Therefore, the present invention provides a novel platform technology that overcomes these problems, minimizes recognition by immune cells in the body, and ensures stable distribution in the body and excretion from the body.

[0039] In order to solve the problem of immune side effects, the present invention provides a non-immunogenic polysaccharide cross-linked colloidal particle platform with a compact spherical three-dimensional network structure that does not cause immune stimulation through interaction with surface receptors of immune cells by selectively modifying the -OH functional group of a monosaccharide with a first cross-linking agent of the epoxide series to chemically cross-link and precisely controlling the cross-linking conditions (e.g., type and amount of cross-linking agent, reaction temperature, reaction time, pH, concentration of reactants, etc.) even when various polysaccharide polymers are cross-linked, thereby preventing immune stimulation.

[0040] In particular, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention having a compact spherical three-dimensional network structure are designed / synthesized so that they have a weak binding affinity to surface receptors of immune cells (e.g., pattern recognition receptors (PRRs), BCRs) when distributed in the body, and thus do not serve as external stimuli that turn on an immune response switch (immune cell activation signal), and thus do not induce B cell activation and antibody production.

[0041] The present invention is based on the premise that when various polysaccharide polymers such as dextran are chemically cross-linked, subtle differences in the degree of cross-linking, molecular weight distribution, and functional group arrangement occur within a three-dimensional network structure. However, the core of the present invention is that, despite such structural heterogeneity, the surface of a compact, spherical three-dimensional network structure formed by chemically cross-linking by selectively modifying the -OH functional group of a monosaccharide, which is a repeating structure of a polysaccharide to be cross-linked, with a first cross-linking agent of the epoxide series is optimized so as not to interact with pattern recognition receptors (PRRs, e.g., TLRs, C-type lectin receptors) or B cell receptors (BCRs) of immune cells.

[0042] That is, the non-immunogenic polysaccharide cross-linked colloidal particle of the present invention is characterized in that it selectively modifies the -OH functional group of a monosaccharide with a first cross-linking agent of the epoxide series to provide a compact spherical three-dimensional network structure in which the polysaccharide is chemically cross-linked, 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, which are building blocks of the polysaccharide, are modified by the cross-linking agent, and at this time, the number of amine functional groups derived from the cross-linking agent exposed on the particle surface is controlled, and some or all of the amine functional groups are modified with carboxylic acid (-COOH) to control the surface charge in the range of -20 mV to 0 mV, thereby minimizing recognition by surface receptors of immune cells and suppressing immune activation even upon repeated administration.

[0043] Inhibition of Binding to Immune Cells: When a polysaccharide polymer is cross-linked at the -OH functional group of a monosaccharide according to the present invention to form a compact nanoparticle having a near-spherical shape, the degree of freedom of rotational motion of the chemical bonds is extremely reduced, making it difficult to provide a three-dimensional structure capable of binding to the antigen-binding site of a pattern recognition receptor (PRR), BCR, or antibody through a combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions. Therefore, the binding affinity with PRR and BCR is significantly reduced, thereby blocking signal transduction that induces an immune response.

[0044] Stability upon repeated administration: This structural optimization ensures that repeated administration does not induce immune activation and antibody production (anti-drug antibodies, ADAs), ensuring safety for long-term therapeutic and diagnostic use (e.g., contrast media).

[0045] Therefore, the polysaccharide cross-linked colloidal particles of the present invention effectively block the immunogenic portion of polysaccharides that can act as foreign antigens, thereby inhibiting the activation of immune cells (macrophages, dendritic cells, etc.) and the induction of antibody production. In particular, in the case of B cells, abnormal immune signaling caused by BCR clustering is blocked, significantly reducing the risk of allergic and hypersensitivity reactions.

[0046] In addition, in the polysaccharide cross-linked colloidal particles of the present invention, a robust three-dimensional network formed through chemical cross-linking by selectively modifying the -OH functional group of a monosaccharide with a first cross-linking agent of the epoxide series provides resistance to hydrolysis by enzymes in the body, and the realization of an optimal particle size through control of the degree of cross-linking and the appropriate surface charge control through control of the number of cross-linking agent-derived amine functional groups exposed on the particle surface enable stable excretion from the body through normal metabolic and excretory pathways (e.g., kidney and lymphatic system).

[0047] Therefore, considering the complexity of polysaccharide structures and differences in immune system recognition across species, the polysaccharide cross-linked colloidal particles of the present invention are designed to complement differences in metabolic stability and clearance rates in animal models and humans. This reduces the risk of unexpected drug accumulation or the development of toxic metabolites in human clinical trials.

[0048] In short, the non-immunogenic polysaccharide cross-linked colloidal particle platform technology of the present invention minimizes the structural heterogeneity that may arise from the formation of a three-dimensional network due to cross-linking of polysaccharides by selectively modifying the -OH functional groups of monosaccharides with a first cross-linking agent of the epoxide series to chemically cross-link the polysaccharide into a compact, spherical three-dimensional network structure, preferably a three-dimensional network structure in which at least 60%, at least 70%, at least 90%, or at least 95% of the total number of monosaccharides, which are building blocks of the polysaccharide, are modified by the cross-linking agent, inhibits degradation by body enzymes, and further minimizes interaction with immune cells through surface modification technology, enabling stable excretion from the body. In addition, it can effectively prevent immune responses and side effects caused by foreign substances by compensating for differences in immune recognition between species and variability in metabolic pathways.

[0049] Therefore, the polysaccharide cross-linked colloidal particles of the present invention can be applied to early diagnosis of inflammatory diseases, targeted drug delivery, and the construction of safe drug delivery systems. In particular, by minimizing adverse effects associated with the body's immune response (e.g., allergies, inflammation, hypersensitivity, edema), they can effectively address the issue of reduced therapeutic efficacy observed in existing systems.

[0050] The polysaccharide cross-linked colloidal particle-based T1 MRI contrast agent of the present invention was confirmed to move to the central lymphatic vessel without remaining in the lymph node where various immune cells gather due to interaction with immune cells (Examples 5-7) from MRI images (Fig. 18), and the physicochemical and structural properties realized through the method for producing the polysaccharide cross-linked colloidal particles of the present invention were elucidated for the first time, thereby completing the present invention.

[0051] In addition, in preclinical animal studies, it was confirmed that there was no toxicity even with repeated administration, as long as gelation or aggregation did not occur at the administration site through dose adjustment (NOAEL for repeated administration in Table 4 and Table 7).

[0052] Furthermore, it was observed through clinical trials and animal experiments that the polysaccharide cross-linked colloidal particles of the present invention do not cause an immune response (e.g., an immune side effect) in the immune system when administered to the body and are discharged through the lymphatic system (drainage) and circulated through the bloodstream and then excreted in urine through the kidneys, and are not hydrolyzed by glycoside hydrolases distributed in the body, so that potential immunogenic sites are not exposed.

[0053] The polysaccharide cross-linked colloidal particle according to the present invention is an amorphous water-soluble colloidal particle formed by (i) modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide, with a first cross-linking agent having an epoxide group in an aqueous solvent, and (a) directly cross-linking between a functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group and / or (b) cross-linking of two spatially adjacent functional groups modified with the first cross-linking agent through a second cross-linking agent having two or more amine groups (-NH2) and / or (ii) controlling the number of basic amine groups derived from the cross-linking agent exposed on the surface through modification with a -COOH functional group.

[0054] At this time, the polysaccharide cross-linked colloidal particles, in which the -OH functional group of the polysaccharide to be cross-linked and dispersed in an aqueous solvent according to the present invention is modified with a cross-linking agent and the surface charge of the particles is controlled within a range of -20 mV to 0 mV through the -COOH functional group exposed on the surface, may have a hydration diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and even more preferably 6 nm or less.

[0055] According to the present invention, a polysaccharide cross-linked colloidal particle in which the -OH functional group of the polysaccharide to be cross-linked and dispersed in an aqueous solvent is modified with a cross-linking agent is

[0056] When 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of polysaccharides, are modified by a cross-linking agent, the hydration diameter is 2 to 10 nm, and the surface charge of the particle is controlled within the range of -20 mV to 0 mV through the -COOH functional group exposed on the surface,

[0057] (a) It is filtered in the renal capillaries but does not penetrate the walls of healthy capillaries (normal capillaries), (b) It is excreted only into the distal lymphatic vessels without venous contamination depending on the site of administration, and (c) It has reduced or minimized immunogenicity against immune cells compared to the cross-linking target polysaccharide, and is not hydrolyzed by body enzymes, so it is excreted from the body without exposing potential saccharide-based immunogenic sites (Examples 3, 4, 5, and 6).

[0058] In addition, the present inventors discovered that when complex branched dextran or a derivative thereof is crosslinked with a crosslinking agent at the -OH functional group of a glucose building block in an aqueous solution, one to three dextran or a derivative polymers form spherical nanoparticles through intramolecular and / or intermolecular crosslinking, and that such dextran crosslinked-based nanoparticles exhibit excellent colloidal stability without aggregation along with compressible properties, and confirmed that the functional groups derived from the crosslinking agent exposed on the surface of the dextran crosslinked-based nanoparticles can be used to modify the surface of functional nanoparticles for in vivo introduction and drugs (Examples 1 and 2). At this time, by controlling the type and / or degree of substituents that substitute the cross-linking agent-derived functional groups exposed on the surface of the dextran cross-linked product-based nanoparticles, not only could the surface charge be freely controlled (Examples 1-5 and 1-6), but also the number of functional molecules (e.g., drugs) bound to the dextran cross-linked product-based nanoparticles could be controlled by controlling the reaction ratio with the drug (Example 1-7). In addition, since the molecular weight of dextran used in the synthesis of the dextran cross-linked product-based nanoparticles used as a modifier increased, the hydrodynamic size of the dextran cross-linked product-based nanoparticles increased, it was confirmed that it was possible to control the hydrodynamic size of the in vivo injection complex modified with the dextran cross-linked product-based nanoparticles.

[0059] In addition, we confirmed the squeezability characteristics of cross-linked dextran nanoparticles (C-DNP) compared to inorganic nanoparticles to suit the in vivo biological environment, i.e., biological microstructures, which are the target of the passage route; the squeezability of iron oxide nanoparticles coated with C-DNP and the resulting diffusion and mobility in biological tissues were also confirmed. In addition, the results of synthesizing and analyzing iron oxide nanoparticles coated with C-DNP of various molecular weights showed that the hydrodynamic size increases as the molecular weight of dextran used in the synthesis of C-DNP increases, enabling size control; that the surface charge of iron oxide nanoparticles coated with C-DNP can be controlled by controlling the surface charge of C-DNP; and that C-DNP-coated iron oxide nanoparticles have excellent colloidal stability because they can be dispersed for a long time without precipitation due to aggregation under various physiological conditions (salt, pH). We found that when compressible C-DNP with functional groups derived from coordination-capable cross-linking agents exposed on the surface is used, iron oxide nanoparticles can be coated with 20 times less amount than when using uncross-linked dextran, and excellent colloidal stability can be secured; that in the case of C-DNP-coated iron oxide nanoparticles, the anti-opsonization effect can be controlled by controlling the surface charge of the nanoparticles; and that the pharmacodynamic behavior of C-DNP-coated iron oxide nanoparticles can be controlled by controlling the size and surface charge of C-DNP.

[0060] As described above, the polysaccharide cross-linked colloidal particles of the present invention have been experimentally proven to be a non-immunogenic platform that can effectively avoid immune-induced inflammatory responses (e.g., pain, redness, swelling, etc.) commonly observed in existing foreign substance-based drugs. When cross-linked dextran (C-DNP) was intravenously administered to rats at the same dose (125, 250 mg / kg) as dextran T-10 before cross-linking, acute swelling in the face and limbs was observed only in the dextran-administered group, whereas the polysaccharide colloidal particles with a high cross-linking rate of the present invention (NEMO-103, cross-linking rate ≥60%) did not exhibit any side effects under the same conditions (see Example 2-1 and Fig. 2). This suggests that the cross-linking shields the immune recognition structure on the polysaccharide surface, thereby inhibiting the interaction with immune cell receptors.

[0061] In addition, the results of the evaluation of NF-κB activation, a key signaling factor in inflammation and immune responses, confirmed that the cross-linked particles of the present invention did not significantly stimulate the NF-κB transcription pathway in macrophages despite stimulation by PAMPs (Figs. 4 and 6). This demonstrates that the particles according to the present invention have biocompatibility that allows them to be stably used in the body without stimulation of the immune system.

[0062] In addition, in Example 2, which utilized the polysaccharide cross-linked colloid of the present invention as a drug delivery vehicle, a drug conjugate was produced in which an immuno-oncology small molecule ligand, such as a TLR 7 / 8 agonist, was evenly distributed on the surface, and it was confirmed in vitro and in vivo experiments that these effectively induced intracellular signal transduction and immune cell activation by specifically binding to cell membrane receptors (see Figs. 4, 5, 6, and 7). These results demonstrate that the present invention can simultaneously implement the immune evasion property and drug delivery ability of a polysaccharide-based biomaterial through a highly controlled cross-linking reaction.

[0063] In Example 4, NEMO-103, a T1 contrast agent based on a dextran crosslinker, was synthesized, and its physicochemical properties and imaging efficacy in a phase 1 / 2a clinical trial were evaluated. NEMO-103 was applied to shoulder joint MRI and secured image quality (based on CNR, swelling, and Turing test) equivalent to or higher than that of the control group (GBCA-based contrast agent), and suggested the possibility of extending the imaging time through improved reabsorption resistance of the contrast agent. In addition, the contrast agent of the present invention was confirmed to be stable without aggregation as a result of a colloidal stability evaluation for 28 days under physiological conditions of pH 5 to 9 and NaCl up to 1,000 mM.

[0064] To achieve lymphatic imaging without venous contamination, Example 5 evaluated the applicability of INV-001, an iron-based T1 contrast agent, to various animal MRL (magnetic resonance lymphangiography). INV-001 is a structure in which iron ions are coordinated to dextran cross-linked colloids. Because its hydrodynamic size (~4 nm) is smaller than the renal filtration limit, it is not absorbed into the venous system when injected intradermally, but is selectively absorbed and excreted through the lymphatic system. Sprague-Dawley rats (250-300 g) were injected intradermally with INV-001 in both hindlimbs, and 3D TOF sequence imaging was performed using a 9.4T MRI machine. As a result, no venous contamination was observed in the INV-001-administered group compared to Gd-DOTA. In particular, INV-001 showed maximum enhancement between 16 and 32 minutes after injection for the popliteal lymph nodes and lymphatic vessels, and clear lymphatic images were obtained without interstitial enhancement or lymph node enlargement even in the range of 0.45 to 0.75 μmol.

[0065] In Example 5, which involved healthy beagle dogs, the optimal concentration and dose for MRL application of INV-001 were derived. INV-001, a nano-contrast agent with a dextran core-iron oxide shell structure, selectively imaged only lymphatic vessels and lymph nodes when injected intradermally at a concentration of 15 mM and a dose of 0.056 and 0.112 mg Fe / kg, respectively, with no venous contamination observed. This demonstrates that INV-001 provides equivalent or better lymphatic system image quality even at significantly lower concentrations (0.067 and 0.13 mL / kg) compared to existing GBCA-based contrast agents (typical concentrations of 500-1,000 mmol / L and doses of 0.1-0.2 mL / kg). These results contribute to the establishment of preclinical MRL dosing parameters for INV-001 and support the potential for clinical expansion of the development of lymphoselective contrast agents.

[0066] Example 6 shows that INV-001 is a lymphatic system-specific T1 MRI contrast agent with excellent safety based on immunoinactivation, demonstrating stable specific contrast effects on lymph nodes and lymphatic vessels in a phase 1 clinical trial, while also demonstrating excellent safety based on non-immunoactivation, including non-MTD achievement, absence of ADR and SAE, and absence of local inflammatory reaction even in tissues densely populated with immune cells.

[0067] Meanwhile, in Example 3, it was confirmed that the dextran crosslinked nanoparticles formed by intramolecular and / or intermolecular crosslinking of 1 to 3 dextran or dextran derivatives used in Example 1 with a crosslinking agent at the -OH functional group of the glucose building block can be expanded into polysaccharide crosslinked colloidal particles formed by intramolecular and / or intermolecular crosslinking of 1 to 3 linear polysaccharides or 2 to 30 cyclic polysaccharides, which are crosslinking targets, dispersed in an aqueous solvent, with a crosslinking agent at the -OH functional group of the monosaccharides, which are building blocks thereof.

[0068] That is, the polysaccharide cross-linked colloidal particles of the present invention, in which (i) the -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-linking agent having an epoxide group in an aqueous solvent, (a) the functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group are cross-linked intramolecularly and / or intermolecularly through a second cross-linking agent having two or more amine groups (-NH2), and (ii) the number of basic amine groups derived from the cross-linking agent exposed on the surface is controlled through modification with -COOH functional groups, have a hydration diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and even more preferably 6 nm or less, and the surface charge of the particles is controlled within a range of -20 mV to 0 mV through the -COOH functional groups exposed on the surface. Polysaccharide cross-linked colloidal particles can be provided, and through Example 3 and FIGS. 8 to 10, it was confirmed that after intravenous injection, polysaccharide cross-linked colloidal particles enter the blood vessels of the nephron and are excreted as urine through the filtration mechanism of the kidney (as confirmed by MRI signals in the bladder), and while they are excreted as urine through the kidney after circulating in the blood vessels, their immunogenicity against immune cells in the liver / spleen immune system (e.g., phagocytosis by macrophages, recognition by B cells) is minimized, and they are not hydrolyzed by enzymes in the body.

[0069] In short, when 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of the polysaccharide, are modified by a cross-linking agent according to the present invention, the polysaccharide cross-linked colloidal particle has at least one -OH functional group in at least one of two consecutive monosaccharides in the polysaccharide chain mostly modified by the cross-linking agent, so that not only is the immunogenicity for immune cells reduced or minimized compared to the polysaccharide to be cross-linked, but it is also excreted from the body without being hydrolyzed by enzymes in the body.

[0070] At this time, the monosaccharide in the polysaccharide chain may have two to three -OH functional groups, and the monosaccharide modified with a cross-linking agent may have one of the two to three -OH functional groups modified with a cross-linking agent.

[0071] In terms of reducing or minimizing immunogenicity to immune cells compared to the polysaccharide to be cross-linked without being hydrolyzed by enzymes in the body so that it can be excreted from the body without exposing potential saccharide-based immunogenic sites, the greater the number of monosaccharides modified by the cross-linking agent among the total number of monosaccharides in the polysaccharide chain, the better.

[0072] Meanwhile, the polysaccharide cross-linked colloidal particles 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, which are building blocks of polysaccharides, are modified by a cross-linking agent and the surface charge is controlled within the range of -20 mV to 0 mV through the -COOH functional groups exposed on the surface,

[0073] In an aqueous solvent, (i) a -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-linking agent having an epoxide group, (a) directly between a functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group and / or (b) two spatially adjacent functional groups modified with the first cross-linking agent are cross-linked intramolecularly and / or intermolecularly via a second cross-linking agent having two or more amine groups (-NH2), thereby forming a polysaccharide cross-linked particle, and (ii) controlling the number of basic amine groups derived from the cross-linking agent exposed on the surface by modification with a -COOH functional group.

[0074] At this time, the polysaccharide cross-linked colloidal particles of the present invention can be designed so that 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of the polysaccharide, are modified by the cross-linking agent while having a hydration diameter of 2 to 20 nm by controlling the molecular weight of the polysaccharide, the length of the polysaccharide main chain, the type of cross-linking agent during cross-linking, the amount and administration speed of the cross-linking agent administered during the synthesis reaction, and the modification of additional chemical functional groups, and the intramolecular cross-linking density and / or the intermolecular cross-linking density can be controlled to be high so as to minimize adverse immune reactions in the human body.

[0075] For example, the present invention can provide polysaccharide cross-linked colloidal particles that (a) are filtered in the capillaries of the kidney but do not penetrate the walls of normal capillaries, (b) have a hydrated diameter of 2 to 10 nm, preferably 8 nm or less, more preferably 6 nm or less, and a surface charge of -20 mV to 0 mV so that they are selectively excreted only into the distal lymphatic vessels without penetrating or excreting into the capillaries at the injection site, and (c) are not hydrolyzed by enzymes in the body so that immunogenicity to immune cells is minimized and they are excreted from the body without exposing potential saccharide-based immunogenic sites, and exhibit pharmacokinetics (absorption, distribution, metabolism, and excretion modes) and pharmacodynamics (effects on the body) that are completely different from those of conventional nano-drugs with different physicochemical properties or polysaccharide cross-linked colloidal particles that do not satisfy all of the above conditions (a), (b), and (c). In addition, the polysaccharide cross-linked colloidal particles according to the present invention have changed biological properties, such as reduced immunogenicity against immune cells, and thus can be used in different dosages, dosage forms, and / or administration routes than conventional nano-drugs with different physicochemical properties.

[0076] Non-limiting examples of crosslinking agent-derived functional groups or hydrophilic functional groups exposed on the surface of the polysaccharide crosslinked colloidal particles of the present invention include amine groups, carboxyl groups, hydroxyl groups, and / or thiol groups.

[0077] The -OH functional group and / or cross-linking agent-derived functional group of monosaccharides, which are building blocks of polysaccharides, can be modified through various covalent linkages as shown in Table 1 below.

[0078]

[0079] Reactive functional groups such as amine, thiol, carboxyl, and hydroxyl facilitate not only surface modification but also chemical bonding with ligands that specifically bind to receptors of specific cells, antibodies or fragments thereof, proteins, peptides, nucleic acids (DNA, RNA, or fragments thereof), biopharmaceuticals, or various types of small molecule drugs.

[0080] The polysaccharide cross-linked colloidal particles of the present invention can be covalently or coordinately bonded to nanoparticles, molecules or metal ions through the amine functional group or -COOH functional group derived from the cross-linking agent exposed on the surface.

[0081] Therefore, the polysaccharide cross-linked colloidal particles according to the present invention can have various and precise controllable physicochemical properties by modifying the cross-linking agent-derived functional groups exposed on the surface, such as amine groups, with -COOH functional groups and / or nanoparticles, molecules or metal ions, thereby enabling precise control of body distribution and excretion.

[0082] In particular, the surface of the polysaccharide cross-linked colloidal particle of the present invention can be covalently or coordinately bound to a functional nanoparticle (e.g., iron oxide nanoparticle, antibody, protein or nucleic acid having a three-dimensional structure), molecule (e.g., low-molecular-weight drug, hormone, neurotransmitter) or metal ion through a cross-linking agent-derived amine functional group or a -COOH functional group.

[0083] Therefore, the polysaccharide cross-linked colloidal particles according to the present invention can be used as MRI contrast agents, drug delivery systems and / or modifiers. For example, the polysaccharide cross-linked colloidal particles of the present invention can bind iron ions (Fe) through cross-linking agent-derived functional groups exposed on their surfaces, such as amine groups and / or -COOH functional groups having a controlled number. 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ions (Mn 2+) can be coordinately bonded (T1 MRI contrast agent) and can be used to modify the surface of functional nanoparticles, hydrophobic drugs, or drugs with short half-lives.

[0084] The polysaccharide cross-linked colloidal particles of the present invention, to which a low-molecular-weight drug, antibody or ligand that binds to a cell surface ligand or receptor expressed in a specific body part is bound, can be distributed (bio-distributed) to a specific body part and, after a certain period of time, the polysaccharide cross-linked colloidal particles can be discharged into the lymphatic vessels.

[0085]

[0086] [MR images and contrast agents]

[0087] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have an iron ion (Fe) on the cross-linking agent-derived amine functional group or -COOH functional group exposed on the surface. 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+ ) or when iron oxide particles are coordinately bonded, it can be used as an MRI contrast agent that enhances the contrast of images, which is a key drug in modern medicine (Examples 1, 3 to 7).

[0088] In addition, the MRI contrast agent based on non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be variously controlled and modified in terms of hydration diameter, surface charge, and / or cross-linker-derived functional groups exposed on the surface to exhibit desired pharmacokinetics (absorption, distribution, metabolism, and excretion), thereby controlling distribution and excretion in the body, thereby enabling diagnosis of deep organs and expansion of new markets (e.g., specialized in musculoskeletal diseases, specialized in lymphatic vascular diseases), and can also be applied to various diseases that cannot be diagnosed with existing contrast agents and that are difficult to observe with medical diagnostic equipment alone.

[0089] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have an iron ion (Fe) on the cross-linking agent-derived amine functional group or -COOH functional group exposed on the surface. 2+ / 3+), gadolinium ion (Gd 3+ ) or manganese ions (Mn 2+ ) is uniformly distributed on the particle surface through coordination bonding, it can be used as a T1 MRI contrast agent that increases the signal intensity of T1-weighted MRI images by enhancing the relaxation rate of nearby water molecule protons and has the effect of brightening the corresponding area in the MRI image.

[0090] In addition, the non-immunogenic polysaccharide cross-linked colloidal particle-based drug of the present invention, which is a drug in itself or to which a drug is separately combined / loaded, has the function of a T1 MRI contrast agent that exhibits a bright signal in MRI images, and has iron ions (Fe 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ions (Mn 2+ ) can be coordinated to the amine functional group or -COOH functional group derived from the cross-linker exposed on the surface, so that the location of the drug based on the polysaccharide cross-linked colloidal particle can be tracked through MRI images after injection into the body.

[0091] Furthermore, the non-immunogenic polysaccharide cross-linked colloidal particle-based T1 MRI contrast agent of the present invention, which has a hydrophobic ligand linked to its surface, can be used as a T1 MRI contrast agent to determine whether a receptor for a specific hydrophobic ligand is expressed on the surface of a specific cell.

[0092] In addition, iron oxide nanoparticles designed to function as T1 or T2 MRI contrast agents can be surface-modified with the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention, thereby making them squeezable so that they can move between structures in the body without aggregation when injected into the body, thereby performing their function as T1 or T2 MRI contrast agents.

[0093] For example, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can have the surface of superparamagnetic iron oxide nanoparticles coordinated to the amine functional group or -COOH functional group derived from the cross-linking agent exposed on the surface, and in this case, can be used as a T2 contrast agent that darkens the corresponding area in an MRI image through magnetic field disturbance in MRI equipment.

[0094] Therefore, the iron ion (Fe) is added to the cross-linking agent-derived amine functional group or -COOH functional group exposed on the surface of the non-immunogenic polysaccharide cross-linked colloidal particle of the present invention. 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+ ) or the polysaccharide cross-linked colloidal particle-based MRI contrast agent of the present invention, in which iron oxide particles are coordinately bonded, is a drug in itself or the polysaccharide cross-linked colloidal particle-based drug of the present invention to which a drug is separately bonded / loaded can function as an MRI contrast agent, so that after injection into a living body, the location of the polysaccharide cross-linked colloidal particle-based drug of the present invention can be tracked through MRI images, and after injection into a living body, it can be confirmed whether the drug remains in the lymph node due to interaction with immune cells in the lymph node where various immune cells gather (Fig. 18), whether it is phagocytosed by macrophages after injection into a living body, whether it is metabolically decomposed, whether it circulates in the blood, whether it is delivered to the substance of cells through capillaries, whether it accumulates in tissues, whether it is excreted into urine through the kidneys, whether it is excreted into feces, whether it is absorbed into the circulatory system after injection into a living body, whether it leaks through the blood vessel wall, whether it can be collected through urine / feces and reused, and whether it is introduced into cells.

[0095] Iron ion (Fe 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ions (Mn 2+) The polysaccharide cross-linked colloidal particles of the present invention, which are coordinately bonded to the cross-linking agent-derived amine functional group or -COOH functional group exposed on the surface, can be designed / synthesized so that the -COOH functional group and / or the cross-linking agent-derived basic amine groups to which the metal ion is not coordinated are exposed on the surface, and thus can simultaneously function as a chelator of the metal ion (Examples 4 and 5). Therefore, there is no need to use a separate chelator.

[0096] The non-immunogenic polysaccharide cross-linked colloidal particles according to the present invention have the following biocompatibility and imaging diagnostic advantages compared to existing MRI contrast agents:

[0097] (1) Immuno-inactivating design: The structure is designed so that it is not recognized by PRRs (e.g., TLRs) and / or BCRs of immune cells in the body, so that immunogenicity is suppressed even with repeated administration.

[0098] (2) High dispersion stability and optimal particle size: Evenly distributed water-soluble polysaccharide cross-linked colloidal particles of approximately 2-8 nm in size extend the residence time in the lesion tissue with minimal immune stimulation, and induce interstitial space enhancement and / or anatomical space distension, thereby ensuring uniformity of contrast signal (Figs. 11 to 14, Fig. 20).

[0099] (3) Increased lesion specificity by tissue: In inflammatory lesions, nonspecific accumulation is suppressed, and in tumor tissue, selective accumulation is possible due to increased vascular permeability (EPR effect, etc.), thus improving image contrast resolution.

[0100]

[0101] [Immune response, immune cells, and immunogenicity]

[0102] Existing drug delivery systems fail to completely eliminate immunogenic side effects, and repeated administrations can increase immune-mediated side effects. Therefore, the present invention aims to overcome these issues by minimizing binding to immune cells and implementing a structure resistant to degradation by body enzymes.

[0103] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are formed by chemically cross-linking the -OH functional groups of monosaccharides, which are repeating structures of various polysaccharide polymers including dextran, by selectively modifying them with a first cross-linking agent of the epoxide series to form a compact spherical three-dimensional network structure. When this happens, the surface characteristics according to the synthesis conditions (e.g., type and amount of cross-linking agent, reaction temperature, reaction time, pH, reactant concentration, etc.) are designed to minimize the binding affinity with PRRs (e.g., TLRs, etc.) and BCRs, so that they are not recognized as foreign antigens through surface receptors of immune cells unless gelation or aggregation occurs at the administration site through dosage adjustment, and immune activation is suppressed even with repeated administration. In particular, in the case of B cells, they are designed so that abnormal immune stimulation does not occur due to signal enhancement by BCR clustering unless gelation or aggregation occurs at the administration site through dosage adjustment.

[0104] An antigen is a molecule that induces an immune response and can include proteins, DNA, RNA, lipids, and polysaccharides.

[0105] Typically, when a foreign substance is introduced into the body, it is recognized by innate immune receptors (PRRs) or B-cell receptors (BCRs), triggering an immune response. This response can lead to rapid clearance of drug delivery vehicles, adverse reactions, and even reduced efficacy of therapeutic agents or contrast agents.

[0106] In particular, B cells have BCRs clustered in specific locations, determining the strength and persistence of the signal. This leads to an abnormal immune stimulus that promotes B cell proliferation, differentiation, and antibody production, potentially leading to allergies, inflammation, and hypersensitivity reactions.

[0107] Meanwhile, polysaccharides can 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 surface of immune cells (especially macrophages and dendritic cells). This action contributes to enhancing T cell and B cell responses to antigens. Specifically, polysaccharides such as dextran can bind to pattern recognition receptors (PRRs) of immune cells and induce immune cell responses such as increased phagocytosis of macrophages, increased MHC-II expression and enhanced antigen presentation ability of dendritic cells, and promotion of cytokine secretion (e.g., IL-6, TNF-α, IL-12, etc.).

[0108] Accordingly, the present invention provides a platform technology based on non-immunogenic polysaccharide cross-linked colloidal particles that are not recognized by immune cells in the body and cause a minimized immune response, and that can induce innate immunity activation through binding to PRR receptors, enhancement of the function of antigen-presenting cells, etc., by selectively modifying the -OH functional group of monosaccharides, which are repeating structures of various polysaccharide polymers including dextran, with a first cross-linking agent of the epoxide series and chemically cross-linking them, thereby designing and synthesizing non-immunogenic polysaccharide cross-linked colloidal particles whose surface is optimized to avoid interaction with PRRs (e.g., TLRs, etc.) or cell receptors (e.g., BCRs) of immune cells and suppress immune activation even upon repeated administration. Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles that are not recognized by immune cells in the body and cause a minimized immune response according to the present invention can be utilized in various medical applications such as early diagnosis of inflammatory diseases, targeted delivery to lesions, and construction of safe drug delivery systems such as vaccines.

[0109] In the non-immunogenic polysaccharide cross-linked colloidal particle of the present invention, (a-1) a compact spherical three-dimensional network structure is formed by selectively modifying the -OH functional group of a monosaccharide, which is a repeating structure of a polysaccharide, with a first cross-linking agent of the epoxide series, (a-2) inducing an intramolecular and / or intermolecular cross-linking reaction directly and / or through a second cross-linking agent (containing two or more -NH₂ functional groups) between the functional groups modified with the first cross-linking agent and spatially adjacent -OH functional groups, and (a-3) modifying some or all of the basic amine functional groups derived from the cross-linking agent exposed on the surface with -COOH functional groups to control the surface charge within the range of -20 mV to 0 mV.

[0110] (i) The binding affinity with the surface receptors (PRR, BCR, etc.) of the immune cells in the body is significantly weakened, so that the immune response switch of the immune cells is not activated, or it acts as a multivalent antigen with two or more epitopes and fails to induce receptor clustering that initiates the signal transduction cascade.

[0111] (ii) This does not induce B cell activation and antibody production,

[0112] (iii) while ensuring stability from hydrolysis by body enzymes,

[0113] (iv) It can be stably excreted from the body without hypersensitivity even in pathological environments such as inflammatory lesions.

[0114] The fact that the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have significantly weakened binding affinity with surface receptors of immune cells can be confirmed from MRI images (Examples 5-7, Fig. 18) in which the polysaccharide cross-linked colloidal particle-based T1 MRI contrast agent of the present invention does not remain in the lymph nodes where various immune cells gather due to interaction with immune cells but moves to the central lymphatic vessels.

[0115] Furthermore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention significantly weaken the binding affinity with surface receptors of immune cells, thereby suppressing immune recognition and activation signals (e.g., NF-κB pathway) for foreign substances (Example 2, Fig. 4). This significantly reduces immune-mediated side effects by preventing B cell activation and antibody production even with repeated administration (minimizing immunogenicity).

[0116] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are chemically cross-linked by selectively modifying the -OH functional group of a monosaccharide, which is a repeating structure of a polysaccharide polymer, with a first cross-linking agent of the epoxide series, thereby reinforcing the particle structure due to the cross-linking reaction, thereby inhibiting hydrolysis by enzymes in the body, and stably excreted from the body through normal physiological excretion pathways (e.g., kidneys and lymphatic system) due to the optimized particle size and surface charge control (enzyme resistance and stable excretion).

[0117] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be utilized in various medical applications such as early diagnosis of inflammatory diseases, lesion target delivery, and construction of a safe drug delivery system, and in particular, repeated administration is possible without side effects due to an immune response.

[0118] The composition based on non-immunogenic polysaccharide cross-linked colloidal particles of the present invention is a water-soluble polymer platform with a three-dimensional network structure designed through chemical selective modification, and has a precisely controlled cross-linking structure to minimize interactions with surface receptors (PRR, BCR, etc.) of immune cells in vivo. Therefore, according to the present invention, by selectively modifying the -OH functional group of a monosaccharide with an epoxide-based first cross-linking agent and optimizing the cross-linking reaction conditions (type and concentration of cross-linking agent, reaction time, temperature, pH, etc.), a non-immunogenic drug delivery platform capable of significantly suppressing the activation of the immune system even upon repeated administration can minimize inconsistent immune responses that occur due to differences in immune receptors between species, even when the same polysaccharide structure is used.

[0119] In fact, the polysaccharide cross-linked colloidal particle-based contrast agent of the present invention did not show any immune-related adverse reactions even when administered into the body as a drug. In a phase 1 / 2a clinical trial (Stage 1 Safety Set: n=9; Stage 2 Safety Set: n=23), after a single intravenous injection of NEMO-103, a dextran cross-linked particle-based T1 contrast agent, no significant changes were observed in markers indicating systemic inflammatory response, hypersensitivity response, or allergic response (e.g., white blood cell count, C-reactive protein, etc.) in hematological analysis over 24 hours (Examples 4-5). This indicates that the contrast agent component can be safely eliminated through physiological excretion routes without unnecessary interaction with the body's immune system.

[0120] In addition, the results of an animal model toxicity test on INV-001, an iron-based lymphatic contrast agent, showed that despite repeated administration to rats at doses up to 1,500 mg / kg based on dextran, no significant toxic reactions were observed in clinical symptoms (edema, weight changes, organ lesions, etc.), hematological values, or histopathological analysis results (Examples 5-8, Table 7). This is important preclinical evidence showing that the non-immunogenic polysaccharide cross-linked colloidal particle-based composition of the present invention can maintain biocompatibility and immunological safety even when administered at high doses.

[0121] In addition, INV-001 demonstrated both lymphatic system-specific imaging efficacy and immune inactivation-based safety by stably visualizing lymph nodes, which are areas densely populated with immune cells, at high resolution in phase 1 clinical trials, while not reaching the maximum tolerated dose (MTD), not generating drug-related adverse reactions (ADRs) or serious adverse events (SAEs), and confirming non-immunogenicity based on low binding affinity to immune cell receptors (BCRs, PRRs, etc.) (Example 6).

[0122] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particle composition of the present invention is effective as a non-immunogenic polymer-based drug delivery platform that can not only precisely control pharmacodynamic behavior in the body but also effectively suppress immune abnormalities such as inflammation, allergy, and hypersensitivity by minimizing interaction with the immune system, and supports the possibility of various biomedical applications including T1 MRI contrast agents.

[0123] An immunogen is a substance capable of eliciting an immune response. An antigen is any substance (usually a foreign substance) that specifically binds to an antibody or T-cell receptor. All immunogens are antigens, but some, such as haptens, are not immunogens.

[0124] Therefore, in the present specification, “reduced or minimized immunogenicity” may mean that the polysaccharide cross-linked colloidal particles of the present invention do not cause an immune response as an antigen, do not generate antibodies against the antigen, are capable of repeated administration, do not cause an inflammatory response, or do not induce a chronic inflammatory disease.

[0125] In the present specification, “polysaccharide cross-linked colloidal particles having reduced or minimized immunogenicity for immune cells compared to the cross-linked polysaccharide” may mean that the polysaccharide cross-linked colloidal particles of the present invention are not recognized as foreign antigens by the immune system when distributed in the body, do not produce antibodies against the polysaccharide cross-linked colloidal particles, do not cause hypersensitivity reactions or immune-mediated adverse effects, do not activate the complement system, do not cause an inflammatory reaction, are not phagocytosed by macrophages upon repeated administration, are not recognized or bound by pattern recognition receptors (PRRs), do not release cytokines, or do not stimulate the NF-κB activation pathway in macrophages.

[0126] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention may not activate or proliferate immune cells such as B cells and mast cells when distributed in the body.

[0127] Normally, when a foreign antigen binds directly to the cell membrane receptor of an immune cell or indirectly through an antibody, the immune cell can be activated through signal transduction.

[0128] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed not to act as immunogens or antigens that directly bind to surface receptors (PRRs, BCRs, etc.) of immune cells or indirectly bind to immune cells or non-immune cells having Fc receptors via antibodies.

[0129] In the present specification, the antibody may be IgM, IgD, IgG, IgA or IgE.

[0130] Binding of a single antibody molecule to an FcR typically results in no response. If a single antibody were to trigger a response, the immune system would be constantly overactivated. Therefore, a response typically requires multiple immune complexes and cross-linking with the Fc receptor.

[0131] Immune cells that possess Fc receptors include B lymphocytes, mast cells, basophils, follicular dendritic cells, macrophages, neutrophils, eosinophils, and natural killer (NK) cells. They are also present on some non-immune cells, where they contribute to immune surveillance and inflammatory responses. Non-immune cells that possess Fc receptors include endothelial cells and epithelial cells.

[0132] Fc receptors on endothelial cells can mediate the transport of immune complexes across cell layers and may play a role in inflammation. Certain epithelial cells express Fc receptors that can participate in immune responses, particularly at mucosal surfaces.

[0133] In the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention, the cross-linked polysaccharide may be a T-cell-independent multivalent antigen having two or more epitopes. On the other hand, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed / synthesized so as not to cause immune side effects when distributed in the body, for example, so as not to induce B cell activation and antibody production, and specifically so as not to induce the production of antibodies that recognize or bind to multivalent antigens having two or more epitopes.

[0134] Most T-independent antigens, such as polysaccharides, contain multiple identical epitopes on each molecule. These multivalent antigens can effectively cross-link many B cell antigen receptors and initiate responses.

[0135] Dextran, the cross-linking target of the non-immunogenic polysaccharide cross-linked colloidal particle according to one embodiment of the present invention, is a large polysaccharide capable of expressing multiple epitopes, and thus can simultaneously bind to multiple BCRs of a single B cell. This results in clustering (cross-linking) of the BCRs.

[0136] Clustering of BCRs initiates a signal transduction cascade within B cells. Some activated B cells become memory B cells. These cells remain in the body and can mount a faster and more powerful response when encountering the same antigen again.

[0137] Therefore, the polysaccharide cross-linked colloidal particles of the present invention are designed / synthesized so as not to cause immune side effects when distributed in the body, specifically so as not to induce the production of antibodies that recognize or bind to multivalent antigens having two or more epitopes. For example, whether the polysaccharide cross-linked colloidal particles of the present invention are multivalent antigens can be confirmed by checking whether, when mixed with an antibody against a polysaccharide (e.g., dextran) that is the cross-linking target, the particles bind to the antibody, and further, whether aggregation occurs due to binding between multivalent antigen antibodies. In addition, whether the number of cytokines and / or lymphocytes associated with proliferation increases when memory B cells are activated after repeated administration of the polysaccharide cross-linked colloidal particles of the present invention can be confirmed.

[0138] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are compactly spherical by selectively modifying the -OH functional group of a monosaccharide, which is a repeating structure of a polysaccharide to be cross-linked, with a first cross-linking agent of the epoxide series and then forming intramolecular and / or intermolecular cross-links, thereby reducing or modifying / eliminating the combination of hydrogen bonds, van der Waals forces, and hydrophobic interactions that are efficient enough to cause immune side effects through interactions with BCRs / antibodies.

[0139] An epitope is a distinct part of an antigen that the immune system recognizes. In the case of polysaccharides, an epitope may consist of a specific arrangement or shape of sugar residues.

[0140] The individual glucose units and their specific arrangement in the dextran molecule are crucial for recognition (Glucose Residues). α-1,6-glycosidic bonds form the backbone of dextran and contribute to its overall shape and epitope presentation. α-1,3-branch points can create unique structural motifs recognized by BCRs.

[0141] Molecular interactions, i.e., binding, involve noncovalent interactions (e.g., hydrogen bonds, van der Waals forces, and hydrophobic interactions) between the CDRs of the BCR and the glucose residues of dextran. The spatial arrangement of glucose units within dextran and the specific conformation of its branches play a crucial role in binding specificity.

[0142] However, when the polysaccharide is chemically cross-linked intramolecularly and / or intermolecularly by selectively modifying the -OH functional group of the monosaccharide, which is its building block, with a first cross-linking agent of the epoxide series according to the present invention to form a compact nanoparticle having a near-spherical shape, the degree of freedom of rotational motion of the chemical bond is extremely low, making it difficult to provide a three-dimensional structure capable of specifically binding to the antigen-binding site of BCR or antibody through a combination of hydrogen bonds, van der Waals forces, and hydrophobic interactions.

[0143] In brief, B-cell receptors (BCRs) and antibodies that recognize dextran recognize dextran through their variable regions, specifically CDRs, which bind to distinct epitopes formed by dextran's glucose residues and specific structural features. This binding involves a combination of hydrogen bonding, van der Waals forces, and hydrophobic interactions, enabling BCRs / antibodies to specifically recognize and bind dextran.

[0144] Therefore, according to the present invention, (i) the -OH functional group of glucose, which is a building block of dextran, which is a cross-linking target, is modified with a first cross-linking agent having an epoxide group, and (a) directly between the functional group modified with the first cross-linking agent and the spatially adjacent -OH functional group and / or (b) two spatially adjacent functional groups modified with the first cross-linking agent are cross-linked intramolecularly and / or intermolecularly through a second cross-linking agent having two or more amine groups (-NH2), thereby forming a polysaccharide cross-linked particle, and (ii) the number of basic amine groups derived from the cross-linking agent exposed on the surface is controlled through modification with the -COOH functional group, thereby transforming the spatial arrangement of glucose units in dextran and / or the specific conformation of its branches, thereby corresponding to antibodies binding to dextran. The combination of hydrogen bonding, van der Waals forces and hydrophobic interactions at epitope sites within dextran can be reduced or modified / eliminated.

[0145] That is, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are compactly spherical by selectively modifying the -OH functional group of a monosaccharide, which is a building block of the polysaccharide, with a first cross-linking agent of the epoxide series and then chemically forming intramolecular and / or intermolecular cross-links, thereby significantly weakening the binding force with surface receptors (PRR, BCR, etc.) of immune cells in the body, thereby preventing the activation of the immune response switch and reducing or minimizing the immunogenicity / antigenicity of the polysaccharide, which is the target of cross-linking.

[0146] As mentioned above, most T-independent antigens, such as polysaccharides, are multivalent, containing multiple identical epitopes on each molecule. Therefore, even if not recognized by helper T lymphocytes, they can effectively cross-link with many B cell antigen receptors and initiate responses. In this case, memory B cells can form, which remain in the body and can respond more quickly and strongly when encountering the same antigen again. Therefore, repeated injections are impossible due to antibody development.

[0147] In contrast, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed and synthesized so as to reduce or modify / eliminate the combination of hydrogen bonds, van der Waals forces and hydrophobic interactions that are efficient enough to cause immune side effects with BCRs by spheroidizing the polysaccharide molecule(s) to be cross-linked through intramolecular and / or intermolecular cross-linking, and preferably so as not to 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, so that repeated administration can be possible without the development of anti-polysaccharide cross-linked colloidal particle antibodies.

[0148] For example, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be designed / synthesized to have low affinity for B cells or antibodies when administered into the body by controlling the hydration diameter of the particles, 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 cross-linking agent, modification of one end of the cross-linking agent exposed on the surface, cross-linking density, etc.

[0149] The polysaccharide cross-linked 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 cross-linking of the BCR complex, and thus, in the safety evaluation of phase 1 / 2a clinical trials (Examples 4-5), the number of lymphocytes (e.g., B cells) did not increase in a blood test 24 hours after administration. In addition, according to the analysis results of hematological tests in animal experiments of a 4-week repeated (once every 2 weeks, a total of 3 times) intradermal administration toxicity study of INV-001 in beagle dogs (Examples 5-8, Table 7) and hematological tests during a phase 1 / 2a clinical trial (Examples 4-5) related to shoulder MR arthrography using NEMO-103, a T1 MRI contrast agent based on dextran cross-linker, it was confirmed that the polysaccharide cross-linked colloidal particles of the present invention themselves did not show any clinically significant abnormal changes in the complete blood cell count, neutrophil count, eosinophil count, basophil count, and monocyte count in the blood after in vivo administration. Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be designed / synthesized to enable repeated administration.

[0150] The non-immunogenic polysaccharide cross-linked colloidal particles according to the present invention are characterized in that they do not induce one or more immunological adverse effects selected from the group consisting of inflammation, edema, allergy, and hypersensitivity reactions when administered into the body.

[0151] Clinical manifestations of hypersensitivity reactions include urticaria (a localized skin reaction with itching and swelling), angioedema (swelling of the deep layers of the skin around the eyes and lips), and anaphylaxis (a severe systemic allergic reaction that may include symptoms such as difficulty breathing, low blood pressure, and shock).

[0152] In Fig. 2, unlike the crosslinking target, Dextran T10, NEMO-103 using crosslinked dextran (Cdex) did not cause an acute edema reaction in vivo.

[0153] According to the analysis results of hematological tests in animal experiments on the toxicity of INV-001 administered intradermally for 4 weeks (once every 2 weeks, a total of 3 times) in beagle dogs (Examples 5-8, Table 7) and hematological tests during a phase 1 / 2a clinical trial (Examples 4-5) on shoulder MR arthrography using NEMO-103, a T1 MRI contrast agent based on dextran cross-linker, it was confirmed that the polysaccharide cross-linked colloidal particles of the present invention themselves did not cause any clinically significant abnormal changes in the complete blood cell count, neutrophil count, eosinophil count, basophil count, and monocyte count in the blood after in vivo administration, and it can be inferred that the polysaccharide cross-linked colloidal particles of the present invention themselves do not cause inflammatory reactions or hypersensitivity reactions in the body.

[0154] In addition, according to Examples 3-12 and FIG. 10, the polysaccharide cross-linked colloid particle-based T1 MRI contrast agent according to the present invention maintains its performance until it is excreted from the body through urine after circulating in the bloodstream by imaging its path and distribution after administration in the body, for example, by combining the results of the hematological examination (Examples 5-8, Table 7) in the animal experiment of the toxicity study of intradermal administration of INV-001 for 4 weeks (once every 2 weeks, a total of 3 times) in a beagle dog and the analysis results of the hematological examination during the phase 1 / 2a clinical trial (Examples 4-5) related to shoulder MR arthrography using NEMO-103, a dextran cross-linked particle-based T1 MRI contrast agent, it can be confirmed that it was not only not recognized as a foreign substance, but also that the body did not cause an inflammatory response to remove it.

[0155] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention may not be recognized or bound by pattern recognition receptors (PRRs), may not release cytokines, or may not stimulate the NF-κB activation pathway in macrophages (Figs. 4 and 6).

[0156] Representative PRRs include Toll-like receptors (TLR), C-type lectin receptors (CLR), retinoic acid-inducible gene-I-like receptors (RLR), nucleotide oligomerization domain-like receptors (NLR), and absent-in-melanoma-like receptors (ALR).

[0157] As shown in Fig. 4, Cdex itself did not stimulate the NF-κB activation pathway when treated with macrophages. As shown in Fig. 6, Cdex itself did not affect cell viability even when treated with high concentrations, and thus it can be inferred that Cdex itself does not activate (cell proliferation, cytokine release) through cell signaling within macrophages.

[0158] Therefore, according to the analysis results of hematological tests in animal experiments of a 4-week repeated (once every 2 weeks, a total of 3 times) intradermal administration toxicity study of INV-001 in beagle dogs (Examples 5-8, Table 7) and hematological tests during a phase 1 / 2a clinical trial (Examples 4-5) related to shoulder MR arthrography using NEMO-103, a T1 MRI contrast agent based on dextran cross-linker, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention themselves showed no clinically significant abnormal changes in the complete blood count, neutrophil count, lymphocyte count, and monocyte count in the blood after in vivo administration, and it can be inferred that the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are not recognized or bound by pattern recognition receptors (PRRs) or do not release cytokines.

[0159] The linear, branched or cyclic polysaccharide to be cross-linked may be a homopolysaccharide or a heteropolysaccharide.

[0160] Non-limiting examples of linear, branched or cyclic polysaccharides include dextran, cyclodextrin, maltodextrin, inulin, etc., as illustrated in FIG. 8.

[0161] 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).

[0162] For renal excretion, the average molecular weight of the dextran or dextran derivative used may be 10,000 Da or less, and the molecular weight of the spherical dextran crosslinked nanoparticles formed by crosslinking dextran molecules with a crosslinking agent may be 90,000 Da or less.

[0163] Non-immunogenic polysaccharide cross-linked colloidal particles according to one embodiment of the present invention are nanoparticles formed by intramolecular and / or intermolecular cross-linking of a complex branched polysaccharide with a cross-linking agent at the -OH functional group of a monosaccharide as a building block in an aqueous solution, wherein the branched polysaccharide can form dimers or trimers through intermolecular cross-linking, and the polysaccharide cross-linked colloidal particles can be formed by controlling intramolecular cross-linking of only one branched polysaccharide molecule with the cross-linking agent. The degree of compression can be controlled by controlling the degree of cross-linking and / or the molecular weight of the branched polysaccharide, and preferably, the colloidal particles can be compact and spherical.

[0164] In particular, polymer aqueous solutions gain viscosity as their concentration increases. The same applies to aqueous solutions containing cross-linked polysaccharide colloidal particles.

[0165] For example, when dextran molecules having an average molecular weight of 10,000 Da or less are crosslinked with a crosslinking agent at the -OH functional group of a glucose building block in an aqueous solution, 1 to 3 dextran molecules form spherical nanoparticles through intramolecular and intermolecular crosslinking. Therefore, the dextran crosslinked-based nanoparticles of the present invention, in which the dextran molecules having an average molecular weight of 10,000 Da or less are used, have a molecular weight of 90,000 Da or less, and the modified in vivo injection complex can be implemented as a nanostructure having a hydrated diameter of 10 nm or less, preferably 5 nm or less, for renal excretion. Typically, the hydrated diameter of the nanostructure must be 6 to 8 nm or less to enable natural excretion through the kidney.

[0166] Therefore, by designing various methods for synthesizing non-immunogenic polysaccharide cross-linked colloidal particles including dextran cross-linked nanoparticles and modifying them, it is possible to control the distribution and excretion of functional nanoparticles or drugs as modified targets without immune rejection and toxicity, and in some cases, even if they are excreted from the body together with the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention, they are stable without aggregation in plasma and are not metabolized or decomposed in the body after in vivo injection, so that they can be collected and reused through urine or feces. In some cases, the drug can be separated and excreted from the body from the non-immunogenic polysaccharide cross-linked colloidal particles and released from the body through different routes, and in this case, the separated non-immunogenic polysaccharide cross-linked colloidal particles are not metabolized or decomposed in the body, so that they can be collected and reused through urine or feces. For example, the polysaccharide cross-linked colloidal particles isolated from the non-immunogenic polysaccharide cross-linked colloidal particle-based drug of the present invention after in vivo injection can be designed to be absorbed into the blood circulation and excreted through the kidneys into the urine without extravasation through the blood vessel walls.

[0167]

[0168] [Non-immunogenic polysaccharide cross-linked colloidal particles in which more than 60% of the total number of monosaccharides as building blocks are modified by a cross-linking agent]

[0169] The three-dimensional network formed when polysaccharides are chemically cross-linked exhibits subtle differences in the degree of cross-linking, molecular weight distribution, and functional group arrangement, depending on the synthesis conditions (e.g., type and amount of cross-linking agent, reaction temperature, reaction time, pH, reactant concentration, etc.). These differences can alter solubility, stability, and in vivo interactions. Furthermore, particle size, surface charge, and porosity directly influence the biodistribution, in vivo retention time, and even immunogenicity of drugs based on cross-linked polysaccharide colloidal particles. Differences in binding to cell receptors and tissue distribution depending on the degree of cross-linking must be considered.

[0170] The innate immune system consists of cells that nonspecifically recognize and rapidly respond to pathogens. These cells include macrophages, dendritic cells, neutrophils, and natural killer (NK) cells. These cells express distinct pattern recognition receptors (PRRs) to provide initial defense against pathogens. These receptors recognize glycoproteins present on the surface of pathogens and can induce cell activation and inflammatory responses.

[0171] The immune system recognizes repetitive polysaccharide structures to induce an immune response. Therefore, according to the present invention, a highly chemically cross-linked, non-immunogenic polysaccharide colloidal particle-based drug selectively modifies the -OH functional groups of monosaccharides, which are repeating structures of polysaccharides, with an epoxide-based first cross-linking agent to form a highly dense three-dimensional network, thereby controlling structural repeatability and functional group exposure, thereby blocking recognition by pattern recognition receptors (PRRs).

[0172] Representative pattern recognition receptors (PRRs) include Toll-like receptor 4 (TLR4), which recognizes microbial polysaccharides such as lipopolysaccharide (LPS), Mannose receptor (MR), which recognizes mannose structures derived from pathogens, and C-type lectin receptor (CLR), which is a cell membrane receptor that recognizes polysaccharide structures and plays a central role in the response to fungi and some viruses. The present invention prevents activation by immune cells by limiting the exposure of repetitive sugar structures that can be recognized by these PRRs through cross-linking control.

[0173] For B cells to be activated, the receptor (BCR) must be sufficiently cross-linked. However, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have a structural limitation that they cannot induce cross-linking by the B cell receptor (BCR) due to a highly chemically cross-linked structure by selectively modifying the -OH functional groups of the monosaccharides, which are the repeating structure of the polysaccharide, with an epoxide-based first cross-linking agent. This means that even with repeated administration, they do not induce B cell clustering or memory B cell formation, significantly reducing the risk of long-term immune responses or antibody production. In other words, the cross-linked structure of the present invention is designed not to be recognized as a multivalent antigen and to have non-immunostimulatory properties.

[0174] According to the present invention, the -OH functional group of a polysaccharide is selectively modified with a first cross-linking agent of the epoxide series to achieve highly dense cross-linking, thereby limiting the spatial arrangement (accessibility) of repetitive sugar structures and inhibiting the exposure of immune recognition epitopes on the surface of the polysaccharide, thereby significantly reducing the binding affinity with surface receptors of immune cells such as PRRs (e.g., TLRs, C-type lectin receptors, mannose receptors, etc.) and BCRs, thereby effectively blocking immune recognition by surface receptors of immune cells. As a result, the non-immunogenic particle of the present invention is not recognized as a multivalent antigen having two or more epitopes, and does not induce clustering of B cell receptors and formation of memory B cells, so that an immune response is not induced even upon repeated administration.

[0175] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are characterized in that 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of the polysaccharide, are modified by a cross-linking agent. Due to this, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can not only form a compact spherical shape, but also efficiently reduce or minimize immunogenicity to immune cells compared to the polysaccharide as a cross-linking target. In addition, since they are not hydrolyzed by enzymes in the body, immunogenicity due to areas exposed due to hydrolysis can also be minimized.

[0176] At this time, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention, in which the -OH of the polysaccharide dispersed in the aqueous solvent is modified with a cross-linking agent, preferably, at least one -OH of at least one monosaccharide among two consecutive monosaccharides in the polysaccharide is mostly modified with a cross-linking agent, thereby not being hydrolyzed by enzymes in the body and reducing or minimizing immunogenicity against immune cells.

[0177] This is because the non-immunogenic polysaccharide cross-linked colloidal particle-based T1 MRI contrast agent formed by (a) directly cross-linking the functional group modified with the first cross-linking agent and the spatially adjacent -OH functional group of the monosaccharide, which is a building block of a linear polysaccharide, a branched polysaccharide or a cyclic polysaccharide, and (b) intramolecularly and / or intermolecularly cross-linking the functional groups modified with the first cross-linking agent and the spatially adjacent -OH functional groups through a second cross-linking agent having two or more amine groups (-NH2) in an aqueous solvent according to the present invention maintains its performance as a T1 MRI contrast agent until it is excreted from the body as urine after circulating in the bloodstream (Example 3-12 and FIG. 10), by imaging its path and distribution after administration in the body, and thus, it can be inferred that it is not hydrolyzed by enzymes in the body. Therefore, it can be inferred that not only can it be excreted from the body without exposure of potential sugar-based immunogenic sites due to hydrolysis, but also the resulting immunogenicity is minimized.

[0178] Specifically, in the animal test (intravenous administration) of Example 3, the phase 1 / 2a clinical trial (musculoskeletal contrast agent) of Examples 4-5, and the animal test (lymphatic contrast agent) of Examples 5-8, it was confirmed through MRI images that the polysaccharide cross-linked colloid particle-based T1 MRI contrast agent of the present invention did not cause an inflammatory reaction when distributed in the body, and was not phagocytosed by macrophages or hydrolyzed by body enzymes, and was excreted in the urine through the kidney.

[0179] The non-immunogenic polysaccharide cross-linked colloidal particle of the present invention is a water-soluble colloidal amorphous nanoparticle in which not only linear polysaccharides (e.g., inulin) dispersed in an aqueous solvent but also complex branched polysaccharides (e.g., dextran) or cyclic polysaccharides (e.g., cyclodextrin) dispersed in an aqueous solvent are cross-linked intramolecularly and / or intermolecularly to form a more complex three-dimensional network through a cross-linking agent, but as described above, 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of the polysaccharide dispersed in an aqueous solvent, are modified by the cross-linking agent, or at least one -OH functional group of at least one monosaccharide among two consecutive monosaccharides in the polysaccharide chain is mostly modified by the cross-linking agent, so that (i) it is not hydrolyzed by enzymes in the body and (ii) the binding force with the surface receptors (PRR, BCR, etc.) of immune cells in the body is significantly weakened, thereby inducing an immune response. The switch is not activated, which can minimize immunogenicity to immune cells by preventing B cell activation and antibody production.

[0180] That is, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have more than 50% of the monosaccharides, which are the building blocks of the polysaccharide, modified by a cross-linking agent, so that even if there is a certain degree of interaction with body tissues or body fluids, the binding force with the surface receptors (PRR, BCR, etc.) of the body's immune cells is significantly weakened, ensuring that an unwanted immune response is not induced.

[0181] Preferably, by more precisely controlling the cross-linking density according to the molecular weight of the polysaccharide, it is not only resistant to hydrolysis by enzymes in the body, but also resistant to hydrolysis at the acidic pH of the stomach or intracellular lysosomes.

[0182] The polysaccharide to be cross-linked may be biodegradable and hydrolyzed by enzymes in the body, but the polysaccharide cross-linked colloidal particles of the present invention, in which (i) the -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-linking agent having an epoxide group in an aqueous solvent, (a) the functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group are cross-linked intramolecularly and / or intermolecularly via a second cross-linking agent having two or more amine groups (-NH2), and (ii) the number of basic amine groups derived from the cross-linking agent exposed on the surface is controlled by modification with -COOH functional groups, may not be hydrolyzed by enzymes in the body.

[0183] Non-limiting examples of enzymes found in the body include glycoside hydrolases, which hydrolyze the glycosidic bonds of polysaccharides into simpler sugars.

[0184] According to the present invention, the polysaccharide cross-linked colloidal particles that minimize immunogenicity against immune cells may have significantly weakened binding affinity to surface receptors (PRR, BCR, etc.) of immune cells in the body, so that they are not recognized by immune cells, do not release cytokines, or do not stimulate the NF-κB activation pathway, but are not limited thereto.

[0185] It can be seen that the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are formed by cross-linking not only linear polysaccharides or cyclic polysaccharides but also highly immunogenic complex branched polysaccharides, and when 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of the polysaccharides, are modified by a cross-linking agent, they are not recognized or bound by pattern recognition receptors (PRRs) on the surface of immune cells (Examples 4-2 and 5-7).

[0186] The degree of immunogenicity of the polysaccharide cross-linked colloidal particles of the present invention may vary depending on the degree of cross-linking, the size of the polysaccharide molecules, and specific chemical modifications.

[0187] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can minimize immunogenicity by significantly weakening the binding affinity with pattern recognition receptors (PRRs) on the surface of immune cells in the body in applications such as drug delivery systems by controlling the degree of cross-linking and the structural / chemical properties of the polysaccharide.

[0188] In short, the non-immunogenic polysaccharide cross-linked colloidal particle-based drug of the present invention can minimize immunogenicity against immune cells by significantly weakening the binding affinity with the surface receptors (PRR, BCR, etc.) of immune cells in the body and thereby avoiding immune recognition.

[0189] Accordingly, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed to minimize immunogenicity against immune cells by significantly weakening the binding affinity with surface receptors (PRR, BCR, etc.) of immune cells in the body, such that 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of the polysaccharide, are modified by a cross-linking agent so that -OH in most monosaccharides is modified by a cross-linking agent, and at the same time, the intramolecular cross-linking density and / or the intermolecular cross-linking density are more precisely controlled, thereby minimizing adverse immune reactions in the human body, for example, not causing immediate and non-specific immune activation in the human body.

[0190] At this time, the non-immunogenic polysaccharide cross-linked colloidal particle of the present invention may be a particle in which 1 to 3 linear or branched polysaccharides or 1 to 30 cyclic polysaccharides are intermolecularly cross-linked in an aqueous solvent.

[0191]

[0192] [Polysaccharide cross-linked colloidal particles designed as non-immunostimulatory particles_drug delivery vehicles]

[0193] The present invention provides non-immunostimulatory and biocompatible water-soluble polysaccharide cross-linked colloidal particles for the purpose of minimizing immune responses and related side effects (e.g., inflammation, hypersensitivity, etc.) that may be induced by foreign substances administered into the body.

[0194] The particles of the present invention are formed by selectively modifying the -OH functional group of a polysaccharide with a first cross-linking agent of the epoxide series, and then (a) directly between the functional group modified with the first cross-linking agent and the spatially adjacent -OH functional groups, and / or (b) intramolecular and / or intermolecular cross-linking of two spatially adjacent functional groups modified with the first cross-linking agent with a second cross-linking agent having two or more amine groups (-NH2). Thereafter, some or all of the surface-exposed amine groups (-NH2) are converted to -COOH functional groups, thereby controlling the surface charge of the particles within the range of -20 mV to 0 mV, thereby simultaneously securing evasion of immune cell detection and resistance to enzymatic degradation. Through this design, the colloidal particles of the present invention are not easily hydrolyzed by enzymes in the body, and can be stably excreted from the body without activating immune cells even in a pathological inflammatory environment.

[0195] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed to minimize the possibility of being recognized by pattern recognition receptors (PRRs) expressed by innate immune cells (e.g., macrophages, dendritic cells, neutrophils, etc.). This allows them to selectively reach inflammatory lesions without stimulating immune cells and to be effectively excreted without residual tissue, thereby enhancing their potential use as imaging contrast agents or therapeutic adjuvants (Example 7).

[0196] The colloidal particles of the present invention can extend the therapeutic window by extending the circulation half-life through immune system evasion. This suppresses immune-induced side effects, such as inflammation and hypersensitivity reactions. Targeted drug delivery to specific tissues or cells is possible based on their immune phagocytosis evasion properties.

[0197] The non-immunogenic polysaccharide cross-linked particles of the present invention form a highly stabilized three-dimensional network structure in which at least 60%, at least 70%, at least 90%, or at least 95% of the total number of monosaccharides is modified by a cross-linking agent. According to Example 3, these particles were confirmed to be filtered in the kidneys through the blood vessels of the nephron and excreted in the urine when injected intravenously (confirmed by MRI-based bladder imaging), indicating that they are safely excreted from the body without accumulation in the body.

[0198] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have various biomedical applications, such as drug delivery vehicles, imaging contrast agents, and immune-evading therapeutic adjuvants. They are particularly useful as delivery platforms for inflammatory diseases, autoimmune diseases, or vaccines.

[0199]

[0200] [Non-immunogenic polysaccharide-crosslinked colloidal particles with polysaccharide crosslinking that prevents them from penetrating the walls of healthy capillaries]

[0201] According to the present invention, (i) a monosaccharide which is a building block of a linear polysaccharide, a branched polysaccharide or a cyclic polysaccharide is modified with a first cross-linking agent having an epoxide group in an aqueous solvent, (a) directly between a functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group and / or (b) two spatially adjacent functional groups modified with the first cross-linking agent are cross-linked intramolecularly and / or intermolecularly via a second cross-linking agent having two or more amine groups (-NH2), thereby forming a polysaccharide cross-linked particle, and (ii) the number of basic amine groups derived from the cross-linking agent exposed on the surface is controlled by modification with -COOH functional groups. The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be designed to have a hydrated diameter of 2 to 20 nm, and in this case, they cannot penetrate the walls of normal capillaries. In addition, when the hydrated diameter is 2 to 10 nm, preferably 8 nm or less, more preferably 6 nm or less, (a) it is filtered in the kidney but does not penetrate the walls of normal capillaries and (b) it can be excreted only into the distal lymphatic vessels without optionally penetrating or excreting into the capillaries at the injection site.

[0202] As the molecular weight of the linear polysaccharide, branched polysaccharide, or cyclic polysaccharide used in the synthesis of polysaccharide cross-linked colloidal particles increases, the hydrodynamic size of the polysaccharide cross-linked colloidal particles increases, and therefore, the hydrodynamic size of the polysaccharide cross-linked colloidal particles of the present invention can also be controlled within the range of 2 to 20 nm.

[0203] The polysaccharide cross-linked colloidal particles of the present invention, designed to have a hydrated diameter of 2 to 20 nm so as not to penetrate the walls of healthy capillaries, may be cross-linked using a first cross-linking agent having an epoxide group and a second cross-linking agent having two or more amine groups (-NH2), and may be cross-linked with 1 to 3 linear or branched polysaccharides or 1 to 30 cyclic polysaccharides.

[0204] In order for the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention to (a) be filtered in the kidney but not penetrate the walls of normal capillaries and (b) be excreted only into the distal lymphatic vessels without optionally penetrating or excreting into the capillaries at the injection site, the hydrated diameter should be controlled to be 2 to 10 nm, preferably 8 nm or less, more preferably 6 nm or less.

[0205] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can provide a drug modality (e.g., an imaging modality of a contrast agent) that is released into the peripheral lymphatics when injected into a tissue site rather than intravenously.

[0206] When the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed to have a hydrated diameter of 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, so as to be filtered by the kidney, they can penetrate the capillary walls of the liver and spleen. Therefore, in this case, the capillaries of the liver and spleen are excluded from the healthy capillaries through which the polysaccharide cross-linked colloidal particles of the present invention cannot penetrate.

[0207] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed to have a hydrated diameter of 2 nm or more and -COOH exposed on the surface so that they cannot penetrate the blood vessel walls of healthy capillaries, resulting in a surface charge of -20 mV to 0 mV.

[0208] Healthy capillaries typically measure 5–10 μm in diameter and have thin walls composed of a single layer of endothelial cells, a functional structure that facilitates the selective exchange of oxygen, carbon dioxide, nutrients, and metabolites. Tight junctions between endothelial cells precisely regulate permeability, playing a vital role in maintaining homeostasis between blood and tissues.

[0209] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention, due to their average particle size and physicochemical properties, are unable to penetrate normal capillary endothelial walls. Consequently, tissue penetration is possible only under pathological conditions involving abnormal capillary structural changes. This characteristic provides a useful platform for the early diagnosis and targeted treatment of diseases associated with pathological changes in capillaries, such as relaxation or damage to inter-endothelial cell junctions, inflammatory responses, and hypoxia-induced angiogenesis.

[0210] Specifically, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention do not penetrate normal capillaries after intravenous injection, but exhibit the characteristic of selectively infiltrating them in pathological situations. Therefore, the particles of the present invention can be usefully utilized as an intravenous imaging contrast agent or drug delivery vehicle, and can be applied to diseases such as capillary leak syndrome, diabetic microvascular complications, thrombosis, chronic hypoxia, or pathologic angiogenesis in a tumor environment in which increased capillary permeability is observed.

[0211] In particular, the intratumoral environment is characterized by intratissue hypoxia and abnormal vascular permeability, and the colloidal particles of the present invention can be applied to various purposes such as anticancer agents, immunomodulators, or MRI contrast agents based on their physicochemical properties that can induce tumor-selective accumulation.

[0212] The non-immunogenic polysaccharide cross-linked colloidal particles according to the present invention are designed to selectively infiltrate only diseased tissues without infiltrating normal tissues, and are thus useful as a biomedical platform technology capable of detecting pathological vascular changes in vivo and improving diagnostic and therapeutic efficiency based thereon.

[0213] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have a hydrated diameter of about 2 to 10 nm, preferably 8 nm or less, more preferably 6 nm or less, and are designed to have a surface charge that is controlled in the range of -20 mV to 0 mV, so that they are filtered in the glomerular capillaries of the kidney but do not penetrate the walls of healthy capillaries with a normal continuous endothelium structure (continuous endothelium with tight junctions and basal lamina). This allows the particles of the present invention to be selectively excreted only through the lymphatic vessels and rapidly excreted through urine after tissue infiltration, thereby minimizing organ accumulation and related toxicity in the body (see Examples 3 and 4).

[0214] For example, the particles of the present invention are excreted in the urine through the filtration mechanism of the kidneys after circulating throughout the body through the bloodstream, and thus the excretion time may be 48 hours or less, preferably 24 hours or less. The non-immunogenic water-soluble polysaccharide cross-linked colloidal particles of the present invention are not metabolized or accumulated in the liver, and therefore are not hepatotoxic, and are resistant to phagocytosis by macrophages and hydrolysis by body enzymes (see FIGS. 9 and 10).

[0215] Starting from various linear, branched, or cyclic polysaccharides, particles are formed by selectively modifying the -OH functional groups of monosaccharides via a first cross-linking agent having an epoxide group, followed by intramolecular and / or intermolecular cross-linking reactions between adjacent -OH groups or via a second cross-linking agent containing an amine group (-NH₂). Subsequently, the remaining amine groups exposed on the surface are modified with -COOH functional groups to impart a negative charge, thereby precisely controlling the surface charge between -20 mV and 0 mV (see Examples 1-16 and Example 3).

[0216] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention were confirmed in Examples 5, 6, and 7 to not penetrate healthy vascular endothelium, but to selectively migrate or be excreted only in pathological conditions or the lymphatic circulation, and particularly to be effectively excreted through the distal lymphatic vessels. In addition, it was observed that INV-001 did not remain in the injection site, lymph nodes, or lymphatic vessels at 24 and 48 hours after intravenous administration, and MRI images (Fig. 18) confirmed that it did not remain in the lymph nodes where various immune cells gather, but moved to the central lymphatic vessels through interaction with immune cells (Examples 5-7). This demonstrates rapid excretion and suppression of body accumulation.

[0217] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention offer the possibility of being utilized as a drug for diagnosis and monitoring of lymphatic diseases and imaging based on four biological properties: (i) tissue-selective penetration blocking and lymphatic drainage, (ii) renal-based excretion, (iii) hepatic metabolism evasion, and (iv) body immune evasion and enzymatic stability (Fig. 20).

[0218]

[0219] [Method for producing non-immunogenic polysaccharide cross-linked colloidal particles]

[0220] The non-immunogenic polysaccharide cross-linked colloidal particle of the present invention may be a polysaccharide cross-linked colloidal particle formed by intramolecular and / or intermolecular cross-linking of 1 to 3 linear polysaccharides or branched polysaccharides or 2 to 30 cyclic polysaccharides, which are cross-linking targets, dispersed in an aqueous solvent, with a cross-linking agent at the -OH functional groups of monosaccharides, which are building blocks thereof.

[0221] The polysaccharide cross-linked colloidal particles of the present invention can be provided by a manufacturing method including, but not limited to, the following steps:

[0222] The first step is to prepare an aqueous solution of linear polysaccharides, branched polysaccharides, or cyclic polysaccharides;

[0223] A second step of modifying the -OH functional group of a monosaccharide, which is a building block of a polysaccharide, with the first cross-linking agent by adding dropwise a first cross-linking agent having an epoxide functional group that reacts with an alkaline aqueous solution and the hydroxyl group (-OH) of the polysaccharide;

[0224] A third step of adding a second cross-linking agent having two or more amine groups (-NH2) dropwise so that spatially adjacent functional groups modified with the first cross-linking agent are cross-linked intramolecularly and / or intermolecularly through the second cross-linking agent, thereby generating polysaccharide cross-linked colloidal particles having terminal amine groups derived from the second cross-linking agent on the surface;

[0225] A fourth step of modifying some or all of the terminal amine groups of a polysaccharide cross-linked colloidal particle having a terminal amine group derived from a second cross-linking agent on the surface by administering an organic acid anhydride to the particle to form a carboxylic acid group and / or a carboxylate group; and

[0226] Optionally, the water-dispersible polysaccharide cross-linked colloidal particles prepared in the previous step are added with iron ions (Fe 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+) by administering a precursor (e.g., iron chloride) or an aqueous solution of iron oxide nanoparticles, (i) by introducing iron ions (Fe) to the amine functional groups or -COOH functional groups derived from the cross-linker exposed on the surface. 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+ ) A fifth step of preparing a complex in which the surface of the polysaccharide-crosslinked colloidal particles or (ii) iron oxide nanoparticles is modified with the polysaccharide-crosslinked colloidal particles.

[0227] In order to prevent unintended increase in hydration size due to swelling of linear polysaccharide, branched polysaccharide or cyclic polysaccharide molecules, and to reduce or minimize immunogenicity to immune cells compared to polysaccharides that are cross-linked without being hydrolyzed by enzymes in the body so that they are excreted from the body without exposing potential saccharide-based immunogenic sites, the present invention is a composition in which linear polysaccharides, branched polysaccharides or cyclic polysaccharides are cross-linked intramolecularly and / or intermolecularly in an aqueous solution.

[0228] At this time, it was discovered that when branched polysaccharides such as dextran are cross-linked by a cross-linking agent, 2 to 3 branched polysaccharide molecules form a spherical core through intramolecular and intermolecular cross-linking. Similarly, in the case of linear polysaccharides, 2 to 3 molecules, and in the case of cyclic polysaccharides, up to 30 molecules, can be cross-linked to provide non-immunogenic polysaccharide cross-linked colloidal particles of a hydrated size that cannot penetrate the walls of healthy capillaries (normal capillaries) but can be filtered in renal capillaries through intramolecular and intermolecular cross-linking.

[0229] The non-immunogenic polysaccharide cross-linked colloidal particle of the present invention is a spherical amorphous polysaccharide cross-linked colloidal particle formed by modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide, with a first cross-linking agent having an epoxide group, and (a) directly between a functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group, and / or (b) intramolecular and / or intermolecular cross-linking of two spatially adjacent functional groups modified with the first cross-linking agent through a second cross-linking agent having two or more amine groups (-NH2).

[0230] Non-immunogenic polysaccharide cross-linked colloidal particles can be produced by controlling the number of linear polysaccharide, branched polysaccharide, or cyclic polysaccharide molecules to be cross-linked, either identically or differently, through their synthesis conditions and / or purification.

[0231] For example, the present invention can manufacture non-immunogenic polysaccharide cross-linked colloidal particles in which 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides that are building blocks of the linear polysaccharide, branched polysaccharide, or cyclic polysaccharide is modified by cross-linking with the cross-linking agent by adding (i) a first cross-linking agent having an epoxide group that reacts with a hydroxyl group (-OH) of the polysaccharide and a functional group that chemically bonds with a hydroxyl group (-OH) and / or an amine group (-NH2), and (ii) a second cross-linking agent having two or more amine groups (-NH2), thereby modifying and cross-linking the -OH functional group of monosaccharides that are building blocks of the linear polysaccharide, branched polysaccharide, or cyclic polysaccharide with the cross-linking agent.

[0232] In the second step, the first cross-linking agent having an epoxide group that reacts with a hydroxyl group (-OH) and a functional group that chemically bonds with a hydroxyl group (-OH) and / or an amine group (-NH2) is not limited in type as long as it can modify the -OH functional group portion of the monosaccharide so that (a) a functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group can react directly and / or (b) two functional groups modified with the first cross-linking agent that are spatially adjacent can react with the second cross-linking agent, and preferably, it can be a halo alkyl oxirane, for example, epichlorohydrin.

[0233] In the third step, the second cross-linking agent having two or more amine groups (-NH2) can be replaced with any cross-linking agent that can covalently bond with the functional group derived from the first cross-linking agent that modified the -OH functional group of the monosaccharide, and this also falls within the scope of the present invention.

[0234] In one specific example of the present invention, when epichlorohydrin is used as a first cross-linking agent for modifying the -OH functional group of a monosaccharide, ethylenediamine or diethylenetriamine (DETA) may be used as a second cross-linking agent for participating in cross-linking between spatially adjacent modified functional groups.

[0235] As illustrated in FIGS. 1A and 1B, when a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide is reacted with epichlorohydrin as a first cross-linking agent and ethylenediamine or diethylenetriamine (DETA) as a second cross-linking agent, a series of chemical modifications occur that significantly change the structure and properties of the polysaccharide.

[0236] According to the present invention, when a polysaccharide is modified by using epichlorohydrin as a first cross-linking agent and ethylenediamine or diethylenetriamine (DETA) as a second cross-linking agent in combination, cross-linking increases and stability is improved.

[0237] In particular, the terminal amine groups derived from the second cross-linking agent exposed on the surface of the polysaccharide-cross-linked colloidal particles can impart novel properties, such as enhanced chelating ability, increased reactivity, and the potential for additional functionalization. Therefore, the polysaccharide-cross-linked colloidal particles with terminal amine groups derived from the second cross-linking agent on their surface, generated in the third step, can enhance their ability to interact with other molecules, such as through chelation or drug binding, via the amine groups (-NH2).

[0238] The polysaccharide cross-linked colloidal particles formed in the third step, having terminal amine groups derived from the second cross-linking agent exposed on the surface, can be modified through the fourth step to expose -COOH functional groups on the surface, thereby controlling the surface charge to be -20 mV to 0 mV, and can be made to have a biocompatible pH. In addition, the number of cross-linking agent-derived amine groups exposed on the surface of the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be controlled through a reaction introducing -COOH-containing functional groups.

[0239] For example, when modifying the cross-linking agent-derived amine group exposed on the surface of a polysaccharide cross-linked colloidal particle with a -COOH-containing functional group, the surface charge can be freely controlled within the range of -20 mV to 0 mV by controlling the type and / or degree of modification of the -COOH-containing functional group. In the fourth step, for example, the surface charge can be freely controlled within the range of -20 mV to 0 mV by controlling the amount of succinyl anhydride (SA). As the amount of added SA increases, the surface charge becomes more negative.

[0240] The polysaccharide cross-linked colloidal particles of the present invention have their interactions with water and other molecules changed when the cross-linking agent-derived amine groups exposed on their surface are replaced with carboxyl groups.

[0241] For example, in step 4, the number of cross-linking agent-derived amine groups exposed on the surface of the polysaccharide cross-linked particles of the present invention can be controlled by adjusting the amount of organic acid anhydride administered. Accordingly, the number of functional molecules (e.g., drugs, targeting molecules) bound to the polysaccharide cross-linked colloidal particles can also be controlled by adjusting the reaction ratio with the drug.

[0242] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be covalently or coordinately bonded to functional nanoparticles, molecules or metal ions through cross-linking agent-derived amine groups and / or -COOH functional groups exposed on their surface.

[0243] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can form strong coordination bonds between cross-linking agent-derived functional groups (carboxyl groups or amine groups) and iron oxide nanoparticles, thereby imparting colloidal stability.

[0244] In step 5, iron ions (Fe 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+ ), or non-limiting examples of hydrophilic functional groups that coordinate with iron oxide nanoparticles include hydroxy, carboxylic acid, carboxylate, amine, etc.

[0245] According to the present invention, iron ions (Fe 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+) and non-immunogenic polysaccharide cross-linked colloidal particles can be designed and synthesized to have the function of a T1 MRI contrast agent that exhibits a bright signal in MRI images through steps 1 to 5, so that the location of the polysaccharide cross-linked colloidal particles can be tracked through MRI images after injection into a living body.

[0246] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be designed and synthesized to exhibit a relatively long-lasting contrast effect, thereby extending the scan time during MRI and improving the spatial resolution of MRI, thereby enabling imaging at higher resolution. Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can image lymphatic vessels, which are clinically very important in vivo but difficult to observe with conventional contrast agents, without venous contamination.

[0247] In short, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have cross-linking agent-derived functional groups exposed on their surface and iron ions (Fe 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+ ) can also act as a T1 or T2 MRI contrast agent when coordinated.

[0248] Additionally, metal oxide nanoparticles, such as iron oxide, designed to function as T1 or T2 MRI contrast agents can be surface-modified into non-immunogenic polysaccharide cross-linked colloidal particles in step 5, enabling them to move between structures in the body without aggregation when injected into the body, thereby enabling them to function as T1 or T2 MRI contrast agents.

[0249] Accordingly, since the non-immunogenic polysaccharide cross-linked colloidal particles according to the present invention can function as an MRI contrast agent, the location of the non-immunogenic polysaccharide cross-linked colloidal particle-based drug can be tracked through MRI images after injection into a living body, and it can be confirmed whether it is selectively discharged into peripheral lymphatic vessels without venous contamination, whether it is phagocytosed by macrophages after injection into a living body, whether it is metabolically decomposed, whether it circulates in the blood, whether it is delivered to the parenchyma of cells through capillaries, whether it accumulates in tissues, whether it is excreted into urine through the kidneys, whether it is excreted into feces, whether it is absorbed into the circulatory system after injection into a living body, whether it leaks through the blood vessel wall, whether it can be collected through urine / feces and reused, and whether it is introduced into cells.

[0250] Meanwhile, the non-immunogenic polysaccharide cross-linked colloidal particles formed by intramolecular and / or intermolecular cross-linking of linear polysaccharides, branched polysaccharides, or cyclic polysaccharides with a cross-linking agent according to the present invention can be chemically modified to add, remove, or modify functional groups (-COOH, -NH2, -OH, etc.) so that they are exposed on their surface. Such modifications can be performed to impart new chemical properties or enhance existing properties.

[0251] In the development of drugs or drug carriers based on the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention, both alterations and chemical modifications, which refer to physical or structural changes in the polysaccharide chain, regardless of addition or removal of chemical functional groups, are important.

[0252] Non-immunogenic polysaccharide cross-linked colloidal particles can be optimized for specific applications by adjusting the type and / or molecular weight and / or cross-linking density of linear, branched, or cyclic polysaccharides. Furthermore, chemical modifications can be targeted to introduce new functionality or enhance specific chemical interactions.

[0253] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can precisely control the hydration diameter and surface charge of the polysaccharide cross-linked colloidal particles as desired by controlling at least one of the molecular weight of the polysaccharide, the length of the polysaccharide main chain, the type of cross-linking agent used during cross-linking, the amount and administration speed of the cross-linking agent used during the synthesis reaction, and the modification of additional chemical functional groups, and ultimately can provide the desired blood circulation time and the desired biodistribution and excretion pharmacokinetics.

[0254] In the present invention, the cross-linking agent-derived functional group exposed on the surface of the non-immunogenic polysaccharide cross-linked colloidal particle may be the terminal functional group of the cross-linking agent itself or a modified / substituted functional group thereof. For example, the cross-linking agent-derived functional group exposed on the surface of the polysaccharide cross-linked colloidal particle may be a functional group obtained by modifying / substituting at least a portion of the functional groups of the cross-linking agent exposed on the surface.

[0255] In the present invention, the hydrophilic functional group may be derived from a functional group of a linear polysaccharide, a branched polysaccharide, or a cyclic polysaccharide that did not participate in the cross-linking reaction, a terminal functional group of a cross-linking agent that did not participate in the cross-linking reaction, and / or a functional group obtained by further modifying a terminal of a cross-linking agent exposed after the cross-linking reaction.

[0256] Non-limiting examples of cross-linking agent-derived functional groups or hydrophilic functional groups exposed on the surface of non-immunogenic polysaccharide cross-linked colloidal particles include amine, carboxyl, hydroxyl, and / or thiol groups. Reactive functional groups such as amine, thiol, carboxyl, and hydroxyl not only modify the surface but also facilitate chemical bonding with ligands that specifically bind to receptors of specific cells, antibodies or fragments thereof, antigenic peptides, nucleic acids (e.g., DNA, RNA, or fragments thereof), biopharmaceuticals, or various types of small molecule drugs. In addition, in some cases, the functional groups may be degraded by the acidic environment surrounding the tumor (pH ≤ 7) or by hydrolytic enzymes to release the active drug. For example, non-limiting examples of acid-sensitive bonds that are degraded in the acidic environment surrounding the tumor (pH ≤ 7) include carbonate or ester bonds.

[0257] If the functional group exposed on the surface of the non-immunogenic polysaccharide cross-linked colloidal particle of the present invention has a positive charge such as an amine, cytotoxicity, etc. may occur like other cationic polymers. This can be resolved by replacing some or all of the amine groups with carboxyl groups, methyl groups, ethyl groups, etc.

[0258] Non-limiting examples of hydrophilic functional groups that coordinate with metal ions (e.g., iron ions) include amines, thiols, carboxyls (carboxylates and carboxylic acids), and hydroxyls.

[0259] Furthermore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are amenable to various chemical modifications, such as esterification, etherification, and grafting. These modifications can significantly alter their behavior in various physiological environments.

[0260] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can produce various polysaccharide cross-linked colloidal particle derivatives having unique properties suitable for specific uses through chemical modifications of functional groups such as -COOH, -NH2, and -OH exposed on the surface thereof, and these also fall within the scope of the present invention.

[0261]

[0262] [Non-immunogenic polysaccharide cross-linked colloidal particles with extended injection site retention time and targeting function]

[0263] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be combined with drugs (including contrast agents) or utilized as drug delivery vehicles, and are useful as a means for locally maintaining a high concentration of a drug for therapeutic or diagnostic purposes at a target site. In particular, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have a hydrated diameter of about 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, and a surface charge controlled within the range of -20 mV to 0 mV, thereby being designed to be filtered in the kidney but selectively excreted only through the lymphatic vessels without penetrating normal capillaries.

[0264] Due to these properties, even when not targeted, the particles of the present invention do not rapidly diffuse into the vasculature of the injection site, but rather retain at least 50% of the administered dose at the injection site for at least 1 hour, preferably at least 2 hours, before being drained into the lymphatic vessels. This induces expansion of the interstitial space or anatomical distension, and can function as a filler or space expander (Examples 4 to 7).

[0265] The mechanism of prolonged residence time is interpreted as being due to the large molecular weight and size of the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention compared to single-molecule drugs, and their limited 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 the joint space, intradermally, or subcutaneously, it was confirmed that it was excreted only through the lymphatic vessels, and imaging was maintained for 1 to 2 hours after injection (see Examples 4-6).

[0266] In addition, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be used as a localized drug delivery system to compensate for unfavorable pharmacokinetic / pharmacodynamic (PK / PD) characteristics that may occur when a therapeutic agent rapidly diffuses throughout the body, and can contribute to reducing side effects and improving therapeutic efficacy.

[0267] In addition, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be modified with a ligand, agonist, or antagonist capable of binding to a specific cell surface receptor via an amine group or -COOH functional group on the surface, in which case they can selectively remain at the target site for at least 1 day, preferably at least 4 days, thereby sustaining local action (Example 2-7). In particular, when an antibody or a small molecule ligand is bound, it can specifically bind to the target cell-expressed receptor and then be eliminated from the body through the lymphatic system, thereby simultaneously performing targeting function and biodistribution control.

[0268] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are a technology platform that provides multifunctional advantages such as extended injection site residence time, anatomical structure expansion, lymphatic selective drainage, cell targeting function, suppression of systemic diffusion, and improvement of local diagnostic and therapeutic efficiency, and are thus highly suitable as a diagnostic and therapeutic composition or drug delivery vehicle.

[0269]

[0270] [Targeted contrast agents and targeted drugs / carriers]

[0271] The present invention relates to a method for producing a non-immunogenic polysaccharide cross-linked colloidal particle, comprising: attaching an iron ion (Fe) to an amine group or -COOH functional group derived from a cross-linking agent exposed on the surface of the non-immunogenic polysaccharide cross-linked colloidal particle; 2+ / Fe 3+ ), gadolinium ion (Gd 3+ ), manganese ion (Mn 2+ ) or by coordinating iron oxide particles, a metal complex-based targeting contrast agent or drug carrier platform that can be used as a T1 or T2 contrast agent for magnetic resonance imaging (MRI) is provided.

[0272] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention are designed not to non-specifically bind to cell surface receptors or be absorbed into cells in a non-targeted state, and therefore can act as contrast agents or drug delivery vehicles that selectively accumulate in target cells or tissues by introducing antibodies, peptides, low-molecular-weight ligands, etc. having high affinity for specific cell receptors onto the surface.

[0273] For example, targeted contrast agents can specifically bind to specific tissues or cells, inducing a shortening of the T1 relaxation time, thereby brightening the corresponding area on MR images. Consequently, the distribution or concentration of the targeted biomarker can be inferred from the image. In particular, T1 signal enhancement enables the detection of minute biological changes or pathological signals, offering greater sensitivity and specificity than typical non-targeted contrast agents.

[0274] In addition, because the targeted contrast agent selectively accumulates in a specific area, the contrast agent dose can be reduced, thereby reducing the possibility of side effects. Furthermore, it enables functional and molecular imaging as well as anatomical imaging, thereby providing improved accuracy and information in the diagnosis and pathophysiology evaluation of diseases.

[0275] Representative application examples include cancer detection (high-sensitivity imaging of early tumors and metastatic lesions using antibody-based contrast agents that bind to cancer-specific markers (e.g., HER2, EGFR, etc.), inflammation and infection visualization (detection of infectious lesions using contrast agents conjugated to ligands for inflammatory response-related receptors (TLRs, integrins, etc.) or bacterial / viral surface antigens), and neurological disease diagnosis (precision brain imaging using contrast agents specific for neurotransmitter receptors or structures associated with Alzheimer's disease, such as amyloid plaques and tau proteins).

[0276] Therefore, the non-immunogenic polysaccharide cross-linked colloidal particle-based targeting contrast agent and drug delivery system of the present invention is a highly functional imaging nanoplatform that provides molecular-level disease detection, early diagnosis, targeted treatment evaluation, and imaging possibilities, and is suitable for diagnosis and personalized treatment monitoring of various diseases.

[0277]

[0278] [Uses of non-immunogenic polysaccharide cross-linked colloidal particles]

[0279] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can be designed to enable covalent or coordinate bonding with proteins, peptides, oligonucleotides, small-molecule drugs, metal ions, or nanoparticles by utilizing the amine or carboxyl groups (-COOH) derived from the cross-linking agent exposed on the surface. Accordingly, the non-immunogenic particles of the present invention can be utilized as drug delivery vehicles capable of loading various drugs or functional nanomaterials, or as contrast agents themselves.

[0280] In particular, the non-immunogenic particles of the present invention have a high surface-to-volume ratio, thereby exhibiting high drug loading efficiency per unit mass, and the drug-to-particle ratio (DPR) can be quantitatively and uniformly set through control of surface functional groups. This contributes to improved predictability and repeatability of the pharmacodynamic behavior of the drug delivery system.

[0281] The non-immunogenic particles of the present invention can be precisely controlled in terms of properties such as colloidal stability, surface charge, hydration diameter, compressibility, and immune evasion, thereby having uses such as a colloidal stabilizer for in vivo injection as a drug delivery vehicle, a tissue permeability enhancing or efflux inhibiting controlling agent, a surface charge controlling agent and hydration diameter controlling agent, a surface modifier for avoiding macrophage phagocytosis, a lymphatic or blood circulation half-life controlling agent, a drug distribution or excretion controlling agent, a location tracking contrast agent for MRI imaging, and a nanoplatform for drug loading and release.

[0282] In addition, the non-immunogenic particles of the present invention can provide effects such as minimizing immunogenicity, ensuring the possibility of repeated administration, avoiding antigen exposure, extending blood retention time, and reducing rapid immune clearance by being attached to the surface of a drug or nanoparticle.

[0283] The non-immunogenic particles of the present invention can form a hydration layer through a hydrophilic base structure, thereby preventing adsorption of opsonic proteins and avoiding recognition by macrophages, thereby improving the stability and duration of the drug or particle in vivo.

[0284] In addition, the non-immunogenic particles of the present invention can improve the colloidal stability of insoluble or poorly soluble drugs in vivo, and can be designed as an optimized drug delivery system according to various routes (intravenous, subcutaneous, intramuscular, or oral administration, etc.).

[0285]

[0286] [Non-immunogenic polysaccharide cross-linked colloidal particle-drug complex]

[0287] The present invention provides polysaccharide cross-linked particle-drug conjugates (PDCs) by forming covalent bonds, coordination bonds, hydrogen bonds, or electrostatic bonds with various types of drugs, such as nanoparticles, small molecules, peptides, proteins, and nucleic acids, through amine groups or carboxyl groups (-COOH) exposed on the surface of non-immunogenic polysaccharide cross-linked colloidal particles.

[0288] Drugs are substances for diagnosing, treating, alleviating or preventing diseases, or affecting physiological functions, and in the present invention, they include physiologically active substances such as low-molecular-weight compounds, peptides, proteins (e.g., antibodies), and oligonucleotides (RNA, aptamers). In particular, IC 50 This also includes ultra-toxic payloads with pM levels (e.g., tubulin inhibitors, DNA damage inducers, Top-1 inhibitors, etc.).

[0289] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention can control the number of appropriate functional groups and surface charge according to the type and structure of the drug, and can be used to control metal ions (e.g., Fe) 2+ , Fe 3+ ) or metal oxides (e.g., iron oxide nanoparticles), so it can be used as a surface modifier for functional inorganic particles.

[0290] Specific examples of complexes include payloads conjugated via cleavable linkers, modifiers conjugated to the surface of aggregated hydrophobic drug nanoparticles, and electrostatic complexes of negatively charged nucleic acid-based drugs with positively charged polysaccharide cross-linked colloidal particles.

[0291] In addition, the non-immunogenic polysaccharide cross-linked colloidal particle-drug complex of the present invention has in vivo functionalities such as colloidal stability (maintaining stability in body fluids through formation of a hydration layer by hydrophilic functional groups), pharmacokinetic control (controlling blood circulation time and renal or hepatic clearance rate by controlling hydration diameter and surface charge), immune evasion function (inhibiting phagocytosis and antibody formation by non-immunogenic coating), and targeted drug release (selective drug release in tumor microenvironments, etc., is possible when utilizing an acid-sensitive linker (prodrug strategy)).

[0292] When functional nanoparticles (e.g., superparamagnetic iron oxide, gold, quantum dots, etc.) are coated with the non-immunogenic particles of the present invention, their hydration diameter, surface charge, and biodistribution are controlled, and their effects of evading phagocytosis and extending bloodstream retention time are expected. This increases their potential for use in bioimaging, drug delivery, and as reusable platforms.

[0293] In particular, the PDCs according to the present invention can precisely control the drug-to-particle ratio (DPR), which is crucial for drug delivery efficiency and aggregation stability. The DPR can be controlled within the range of 1 to 10, preferably 1 to 8, more preferably 1 to 6, and even more preferably 1 to 4. For hydrophobic drugs or peptides with concerns about immunogenicity, the range of 1 to 3 is more suitable. The uniformity of the DPR contributes to the predictability of the pharmacokinetics of the drug complex and to the improvement of the repeatability of administration.

[0294] In conclusion, the non-immunogenic polysaccharide cross-linked colloidal particle-drug complex of the present invention has the following technical advantages: applicability to various drugs and functional nanoparticles (flexibility), controllability of body distribution and release (predictability), imparting colloidal stability (physicochemical stability), ensuring suppression of immune response and possibility of repeated administration (clinical safety), and enabling targeted drug release at tumor or infection sites (therapeutic efficiency).

[0295] The non-immunogenic polysaccharide cross-linked colloidal particles of the present invention have a precisely designed surface structure that prevents them from being recognized or activated by immune cells, thereby minimizing immune responses when injected into the body. The particles of the present invention are not easily degraded by body enzymes and can be reliably excreted from the body without hypersensitivity reactions, even in immune-activated pathological environments such as inflammatory lesions.

[0296] In addition, the structural heterogeneity that can occur in the three-dimensional network structure formed by cross-linking of polysaccharides can be controlled by selectively modifying the -OH functional group of monosaccharides with a first cross-linking agent of the epoxide series to chemically cross-link and control the cross-linking conditions and surface modification technology, even when various polysaccharide polymers are used as cross-linking targets, thereby suppressing nonspecific interactions with immune cells and reducing the possibility of side effects due to differences in immune recognition or metabolic pathways between species.

[0297] The non-immunogenic particles of the present invention can precisely control their surface charge within the range of -20 mV to 0 mV, thereby avoiding macrophage phagocytosis, extending circulation time, and controlling excretion from the body. These properties contribute to slowing drug elimination and maintaining blood concentrations, thereby enhancing efficacy and reducing dosage and frequency when utilized as drug delivery platforms, contrast agents, and targeted diagnostic nanoparticles.

[0298] In particular, the polysaccharide cross-linked colloidal particles of the present invention function as non-immunostimulatory nanoparticles, capable of functioning as drugs themselves, drug delivery vehicles, or stealth coating agents. Consequently, adverse effects related to immune activation are suppressed, and the particles can be administered repeatedly without inducing immune tolerance.

[0299] The platform of the present invention also has high applicability as a T1 MRI contrast agent. Designed to drain exclusively into the lymphatic vessels when administered intradermally or subcutaneously, it selectively visualizes only distal lymphatic vessels without venous contamination. This makes it suitable for various clinical applications, including MR Lymphangiography and postoperative tumor imaging, and can provide useful morphological and functional information for planning microsurgical procedures such as lymphovenous anastomosis (LVA). Since it drains exclusively into the lymphatic vessels when administered intra-articularly, it retains the joint space for a longer period of time, compared to existing small-molecule contrast agents that drain into both lymphatic and blood vessels, making it suitable for MR Arthrography.

[0300] The present invention provides key benefits such as minimizing immune responses and ensuring the possibility of repeated administration, extending the circulation time of drugs / contrast agents in the body and precisely controlling their excretion, ensuring resistance to enzymatic degradation in the body and stability in an inflammatory environment, application as a platform technology for precise image diagnosis and lesion target delivery, and enhancing industrial utility in various diagnostic and therapeutic fields.

[0301] Hereinafter, cross-linked dextran structures (nanoparticles) are referred to interchangeably as CDex, DNP, and C-DNP.

[0302] Figure 1 illustrates the structural concept of INV-001 and NEMO-103, their manufacturing method, and their physicochemical properties.

[0303] Figure 2 is a comparison of the in vivo acute edema response of NEMO-103 using crosslinked dextran (Cdex) compared to Dextran T10.

[0304] Figure 3 illustrates the chemical process of conjugating Cdex and Toll-like receptor (TLR) agonist (a), the chemical structure of Cdex@TLR using each conjugation chemistry (b), and the ratio of TLR agonist bound to Cdex using each conjugation chemistry (c).

[0305] Figure 4 is an immunofluorescence staining image of NF-κB p65 signal after 2 hours of treatment in Example 2-5.

[0306] Figure 5 is an immunofluorescence staining image of NF-κB p65 signal after treating cells with TLR agonist for 4 and 6 hours, respectively.

[0307] Figure 6 shows Cdex and TLR A1 agonist, Cdex@TLR A1 and Cdex@TLR B1 After treating the cells for 4 hours, cell viability according to concentration was confirmed using cell counting kit-8 (CCK-8).

[0308] Figure 7: Control group, Doxorubicin, TLR A1 agonist, Cdex@TLR A1 , Cdex@TLR A1 X2 (double dose) and Cdex@TLR B1 This is the experimental design and results of the in vivo efficacy evaluation observed after administration to xenograft model mice.

[0309] Figure 8 is a structural formula of examples of branched polysaccharides or cyclic polysaccharides that are crosslinking targets of polysaccharide crosslinked colloidal particles in Example 3.

[0310] Figure 9 shows a T1 MRI image (a) taken after intravenous administration of a substance synthesized by introducing iron into a crosslinker of Dextran T-5, Maltodextrin, α-cyclodextrin, β-cyclodextrin, and Inulin of Example 3-11, and the signal-to-noise ratio (SNR, b) before and after administration.

[0311] Figure 10 shows a T1 MRI image showing renal excretion of a substance synthesized by introducing iron into the crosslinked form of Dextran T-5, Maltodextrin, α-cyclodextrin, β-cyclodextrin, and Inulin of Example 3-12, after intravenous administration to a mouse.

[0312] Figure 11 shows MRA images taken 30 minutes (a) and 60 minutes (b) after administering NEMO-103 to humans in phase 1 / 2a clinical trials of NEMO-103, and a comparison of the images in terms of CNR (c), Distension (d), and Overall quality (e).

[0313] Figure 12 shows an MRA image taken approximately 30 minutes after administering NEMO-103 strain (a) and gadolinium contrast agent (GBCA, b) to the human body, and a comparison of the images in terms of contrast-to-noise ratio (CNR, c), Distension (d), and Overall quality (e).

[0314] Figure 13 shows MRA images taken 60 minutes after administering NEMO-103 strain (a,c) and GBCA (b,e) to the human body, and a comparison with GBCA in terms of CNR (c), Distension (d), and Overall quality (e) of the images.

[0315] Figure 14 shows the results of evaluating joint swelling, joint boundary clarity, and contrast before and after administering NEMO-103 to humans in a phase 2b clinical trial of NEMO-103.

[0316] Figure 15 compares the effects of Gd-DOTA and INV-001 on the popliteal lymph node (LN) and lymphangiography in Example 5. (a) 3D-TOF scan images before (0 min), 16 min, and 96 min after intradermal injection of Gd-DOTA and INV-001. (b) Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) analysis of the popliteal LN and lymph vessel (LV) after administration of Gd-DOTA and INV-001.

[0317] Figure 16 shows the results of a dose optimization study conducted in Example 5 to improve interstitial space and minimize lymph node enlargement. Visualization of the popliteal LN and LV was performed 16 minutes after injection of various concentrations and doses into the hind limb.

[0318] Figure 17 depicts lymphangiographic images obtained via 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 visualization and the presence or absence of venous contamination after contrast agent injection. INV-001 demonstrates the ability to maintain the contrast effect of lymphatic vessels compared to Gd contrast agents while avoiding venous contamination, and its efficacy is maintained in lymphadenectomy disease models.

[0319] Figure 18 is an image showing that INV-001 injected into the lower extremities of a beagle dog enhances contrast up to the central lymphatic system.

[0320] Figure 19 illustrates a magnetic resonance lymphangiography (MRL) image obtained after injection of INV-001 into a human.

[0321] Figure 20 shows representative images of INV-MRL and NIRF-ICGL at the dorsal (d) and ventral (v) positions. In the control limb, popliteal lymph nodes (LNs) were observed in both modalities (red arrows). Orange arrows indicate areas of diffuse contrast in NIRF-ICGL, where the contrast agent spreads widely over the skin surface, making it difficult to visualize the underlying lymphatic vessels (LVs). However, INV-MRL allows observation of the occult LVs in the same location.

[0322] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended only to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

[0323] Hereinafter, cross-linked dextran structures (nanoparticles) are referred to interchangeably as CDex, DNP, and C-DNP.

[0324] Example 1. Pharmacokinetics of functional nanoparticles coated with polysaccharide cross-linked colloidal particles with controlled size and surface charge.

[0325] 1-1. Synthesis of C-DNP-3 cross-linked with 3 kDa dextran

[0326] 1 g of dextran (molecular weight 3 kDa) was dissolved in 4.2 mL of distilled water, and 8.3 mL of NaOH solution was added. 3.3 mL of epichlorohydrin was added and stirred. 7 mL of diethylenetriamine (DETA) was added and the mixture was further stirred for 24 h to synthesize C-DNP-3, a cross-linked dextran with a molecular weight of 3 kDa. After purification by ultrafiltration, the hydrodynamic size measured by DLS was 3 nm.

[0327] 1-2. Synthesis of C-DNP-5 cross-linked with 5 kDa dextran

[0328] C-DNP-5 cross-linked with dextran having a molecular weight of 5 kDa was synthesized in the same manner 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.

[0329] 1-3. Synthesis of C-DNP-10 cross-linked with 10 kDa dextran

[0330] C-DNP-10 cross-linked with dextran having a molecular weight of 10 kDa was synthesized in the same manner 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.

[0331] 1-4. Synthesis of C-CMDNP-10 cross-linked with 10 kDa carboxymethyl dextran (CM dextran)

[0332] C-CMDNP-10, cross-linked with CM dextran having a molecular weight of 10 kDa, was synthesized in the same manner 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.

[0333] 1-5. Substitution of functional groups exposed on the surface of C-DNP or C-CMDNP

[0334] 1-5-1. Amine group:

[0335] The functional group exposed on the surface of the C-DNP or cross-linked CM dextran nanoparticle (C-CMDNP) synthesized in Examples 1-1 to 1-4 is an amine group.

[0336] 1-5-2. Carboxyl group:

[0337] 30 mg of succinyl anhydride (SA) 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 final C-DNP or C-CMDNP was purified by ultrafiltration.

[0338] 1-5-3. Thiol group:

[0339] N-succinimidyl S-acetylthioacetate was added to 10 mL of C-DNP or C-CMDNP synthesized in Examples 1-1 to 1-4, and stirred for 12 hours. The final C-DNP or C-CMDNP was purified by ultrafiltration.

[0340] 1-5-4. Hydroxide group:

[0341] After adding nitrous acid to 10 mL of C-DNP or C-CMDNP synthesized in Examples 1-1 to 1-4, the mixture was stirred for 12 hours. The final C-DNP or C-CMDNP was purified by ultrafiltration.

[0342] 1-6. Surface charge control of C-DNP / C-CMDNP

[0343] When the amount of SA added according to Example 1-5-2 to 10 mL of C-DNP synthesized in Examples 1-1 to 1-3 is 30 mg, the surface charge is -3 mV. When the amount of added SA 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 charge according to the amount of added SA all showed the same results under the same functional group substitution conditions. When the amount of SA added according to Example 1-5-2 to 10 mL of C-CMDNP-10 synthesized in Example 1-4 with a surface charge of +5 mV is 30 mg, the surface charge is -4 mV. In this way, since the surface charge becomes negative as the amount of added SA increases, the surface charge can be controlled.

[0344] 1-7. Introduction of fluorescent molecules into C-DNP and control of the number of molecules introduced

[0345] 1 mg 5 / 6-carboxyfluorescein succinimidyl ester (NHS-fluorescein) was dissolved in 1 mL of dimethyl sulfoxide (DMSO). 5, 10, and 25 μL of this solution were added to 1 mg of C-DNP-5 (surface charge: +5 mV) exposed to the amine group of Example 1-5-1. After 24 h, C-DNP-5 with fluorescein introduced was purified by ultrafiltration. The number of fluoresceins bound to C-DNP-5 reacted with 5 mmol NHS-fluorescein / DMSO was 4, the number of fluoresceins bound to C-DNP-5 reacted with 10 mmol NHS-fluorescein / DMSO was 9, and the number of fluoresceins bound to C-DNP-5 reacted with 25 mmol NHS-fluorescein / DMSO was 15. As above, it is possible to control the amount of a substance to be bound to C-DNP by controlling the reaction ratio.

[0346]

[0347] Example 2. Polysaccharide cross-linked colloidal particle-drug conjugate (Cdex@TLR as an anticancer agent)

[0348] The cancer cell death mechanism of TLR agonists is described in detail in the literature (Remautet et al., Eur. J. Pharm. Biopharm, 2022, 172, 16.).

[0349] TLR agonists are known to activate various immune cells, including dendritic cells (DCs), macrophages, and T cells. This activation can enhance the body's immune response to cancer cells.

[0350] 2-1. Preparation of nanostructures (Cdex) using dextran T-10

[0351] 180 μmol of dextran T-10 (average molecular weight 10,000 Da) was dissolved in 9 mL of distilled water, and 75 mmol of epichlorohydrin and 75 mmol of NaOH were added. 380 mmol of ethylenediamine was then added and the mixture was stirred at room temperature (RT) for 24 h. This material was then added with 25 mg of anhydrous succinic acid at RT, and after 24 h of succinylation, the mixture was purified by dialysis through a 10 kDa molecular weight cutoff (MWCO) filter. The hydrodynamic size measured by DLS was 5 nm.

[0352] As a result of conducting an edema test by administering NEMO-103 using Cdex, a crosslinked dextran, compared to dextran T-10, which is a crosslinking target, when dextran and Cdex were administered intravenously to rats at the same dose (125, 250 mg / kg), edema was observed in the face and feet after administration only in the case of dextran (Fig. 2).

[0353] 2-2. Introduction of a drug that can bind to Cdex (TLR 7 / 8 agonist)

[0354] Currently, various types of TLR 7 agonists are on the market, and a substance containing an amine group capable of binding to the crosslinker-derived amine group exposed on the surface of Cdex of Example 2-1 was selected. Among these, two types of TLR 7 agonists (TLR A1 , TLR B1 agonist) was selected.

[0355] As illustrated in Fig. 3, Cdex and TLR agonist were conjugated using EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide), NHS (N-Hydroxysuccinimide)-PEG (polyethyleneglycol)-NHS, and BCN (Bicyclo[6.1.0]nonyne) -NHS / N3(azide)-NHS chemical bonding methods (Cdex@TLR, Fig. 3a,b). For chemical bonding using N3-PEG-NHS, 30 mg of N3-PEG-NHS dissolved in 2 mL of DMSO was added to the Cdex material of Example 2-1, and stirred at room temperature for 2 hours. 10 mg of BCN-NHS and TLR A1 After mixing 10 mg of agonist and dissolving it in 2 mL of DMSO, it was added and stirred for 2 hours, and then purified by dialysis using a 10 kDa molecular weight cutoff (MWCO) filter. For chemical bonding using NHS-PEG-NHS, the Cdex material was added to the TLR A1 10 mg of agonist and 20 mg of NHS-PEG-NHS were added, stirred for 2 hours, and purified by dialysis using a 10 kDa molecular weight cutoff (MWCO) filter.

[0356] As a result, TLR bound to Cdex A1The bonding number of the agonist was 3 for the NHS-BCN / N3-NHS chemical bond and 0.45 for the NHS-PEG-NHS chemical bond, and it was confirmed that the NHS-BCN / N3-NHS chemical bond method was the most efficient in terms of chemical bond efficiency (Fig. 3).

[0357] 2-3. Quantification of drugs bound to Cdex

[0358] The substance of Example 2-2 was diluted to 0.5 μg / mL and placed in a cuvette, and the absorbance of the substance was confirmed at a wavelength of 200 to 500 nm using a UV-Vis Spectrophotometer. In the case of Cdex without drug conjugation, there was no absorption at 350 nm, whereas in the case of Cdex with drug conjugation, absorption was confirmed at 350 nm. Through this, the drug was quantified and the amount of drug bound to Cdex was quantified, and TLR per 1 mol of Cdex A1 The number of agonist binding sites was confirmed. At least 3 TLRs per Cdex A1 It was confirmed that the agonist binds.

[0359] 2-4. Hydration diameter and colloidal stability of Cdex@TLR

[0360] TLR in Example 2-2 A1 The agonist-bound Cdex was confirmed to have a hydrodynamic size of 6 nm, and was confirmed to be stably dispersed without agglomeration even in a 1 M NaCl solution, which is a much harsher condition than general physiological conditions.

[0361] 2-5. In vitro efficacy evaluation of Cdex@TLR (confirmation of NF-κB p65 signal)

[0362] Cdex@TLR A1 or Cdex@TLR B1By examining whether the TLR signaling pathway is induced (NF-κB signaling) in cells expressing TLR (Toll-like receptor) during treatment, the efficacy of the polysaccharide cross-linked colloidal particles of the present invention as a drug delivery vehicle can be evaluated.

[0363] To confirm whether the NF-κB signaling pathway was induced through TLR signal activation, the presence or absence of TLR signaling activation according to treatment with Cdex@TLR of Example 2-2 was confirmed using the RAW 264.7 macrophage cell line, a representative immune cell.

[0364] Specifically, the sample Cdex@TLR produced in Example 2-2 A1 and Cdex@TLR B1 Two types, control Cdex, TLR 7 / 8 agonist (TLR A1 , TLR B1 After treating mouse RAW 264.7 macrophage cell line cells with agonist, immunofluorescence staining of NFkB p65 was observed according to the treatment concentration and time presented in Table 2.

[0365]

[0366] When observed through NF-κB p65 immunofluorescence staining after 2 hours of treatment, as shown in Fig. 4, fluorescence was observed throughout the cytoplasm in the control group (cell only, Cdex), and in the TLR 7 / 8 agonist (TLR A1 , TLR B1 agonist) and Cdex@TLR A1 , Cdex@TLR B1In , it was confirmed that NF-κB p65 fluorescence was observed more strongly in the nuclear portion than in the cytoplasmic portion. It is known that when the TLR signal pathway is activated, the fluorescence signal of NF-κB p65 appears more strongly in the nucleus than in the cytoplasm. Cdex@TLR A1 and Cdex@TLR B1 Go TLR A1 , TLR B1 Since the fluorescence intensity by NF-κB p65 is expressed relatively stronger than that of agonist, Cdex@TLR A1 and Cdex@TLR B1 It was confirmed to be effective in TLR signal activation. When the fluorescence intensity according to the treatment concentration was compared, it was observed that the fluorescence signal intensity of NF-κB p65 at 6 μM and the fluorescence signal intensity when treated at 9 μM were similar.

[0367] Among various TLR 7 / 8 agonists, the optimal agonist was selected by checking the degree to which NF-κB p65 expression was observed when cells were treated. Gardiquimod, Resiquimod, TLR A1 , TLR B1 Mouse RAW 264.7 macrophage cells were treated with 6 μM of the agonist for 4 hours. After 4 hours, the cytoplasmic and nuclear fractions of each cell lysate were divided, and the NF-κB p65 signal was evaluated by Western blot. Lamin (nuclear) and β-actin (cytoplasmic) were used as housekeeping proteins for Western blot normalization. As a result of comparing the four agonists, TLR A1 In the case of agonists, the highest expression of NF-κB p65 was observed in the nucleus. Compared to cells treated with the same volume of DMSO, Resiquimod and TLRB1 When treated with an agonist, relatively low NF-κB p65 expression is observed.

[0368] Similar experiments have shown that Gardiquimod, Resiquimod, and TLR A1 , TLR B1 After treating mouse RAW 264.7 macrophage cells with 6 μM of the agonist for 4 and 6 hours, NF-κB p65 expression was observed by immunofluorescence staining (Fig. 5). TLR treated for 4 hours A1 In the case of agonist, it was confirmed that the TLR signaling activation effect was greatest, showing the highest NF-κB 65 expression in the nucleus.

[0369] In short, FIG. 4 suggests that the polysaccharide cross-linked colloidal particles of the present invention as drug carriers provide an orientation that increases the avidity of small molecule drugs, such as TLR 7 / 8 agonists, to cell surface receptors (TLRs) without aggregation, evenly distributed on their surface.

[0370] 2-6. Evaluation of cell viability of TLR-expressing immune cells when treated with Cdex@TLR

[0371] Validated ADC platform Cdex@TLR A1 and Cdex@TLR B1 Two types, Cdex, TLR 7 / 8 agonist (TLR A1 , TLR B1 Cell viability was tested according to the treatment concentration and time of agonist. The cells were evaluated using the mouse RAW 264.7 macrophage cell line expressing TLR.

[0372] Specifically, the ADC platform was divided into untreated, low-concentration, medium-concentration, and high-concentration groups, and then the cell changes according to treatment, concentration, and time were confirmed through the CCK-8 assay, a cell viability assay.

[0373] Looking at the CCK-8 assay results after 4 hours of treatment, it was confirmed that Cdex itself did not change cell viability according to the treatment concentration. TLR A1 The agonist was confirmed to reduce cell viability by approximately 89% starting at 30 μM. Cdex@TLR compared to untreated cells A1 and Cdex@TLR B1 In the case of , cell viability was improved by stimulating the NF-κB activation pathway (Fig. 6). From this, high concentration of TLR A1 While the agonist was aggregated, Cdex@TLR A1 and Cdex@TLR B1 It was found that it activates immune cells expressing TLR without aggregation even at high concentrations.

[0374] 2-7. Drug efficacy in cancer xenograft mice_in vivo efficacy

[0375] The efficacy of the drug was confirmed by monitoring changes in tumor size following drug injection. Cdex@TLR A1 and Cdex@TLR B1 Cancer cell death by administration and subsequent immune response was confirmed in cancer xenograft mice.

[0376] Balb / c nude mice (6 weeks old) were used as experimental animals, and the in vivo efficacy was evaluated by creating a BT-474 breast cancer xenograft mouse model. 1x10 6 BT-474 breast cancer cells and Matrigel were mixed in a 1:1 volume ratio and transplanted in an amount of approximately 100 μL.

[0377] The tumor size is about 50 mm 3 When reached, Cdex@TLR A1 Wow Cdex@TLR B1The efficacy of the drug was confirmed by directly administering the drug to the tumor and measuring changes in tumor size over a two-week period. The test substance was administered on days 0, 4, 7, 12, and 15 (Fig. 7). To ensure effective cancer cell death, doxorubicin was administered to all groups except the control group on days 1 and 9, and a doxorubicin-only group was established separately for comparison (Fig. 7, Table 3).

[0378]

[0379] Compared with the control group (phosphate buffered saline, PBS injection), Doxorubicin, TLR A1 Cdex@TLR compared to the treated group A1 , Cdex@TLR A1 x2 and Cdex@TLR B1 In the treated group, a decrease in relative tumor volume over time was observed.

[0380] Cdex@TLR compared to the control group (PBS injection) A1 , Cdex@TLR A1 x2 and Cdex@TLR B1 The treatment effect was confirmed by observing a decrease in relative tumor volume over time in the treated group. Doxorubicin only and TLR A1 In the agonist treatment group, a slowdown in tumor growth was observed, but no decrease in tumor size was observed.

[0381] In short, the polysaccharide cross-linked colloidal particles of the present invention as drug carriers at tumor sites in the body provide an orientation that increases the affinity of TLR agonists uniformly distributed on their surface to the cell surface receptors (TLRs) of immune cells without aggregation, thereby activating various immune cells such as dendritic cells (DCs), macrophages, and T cells that can enhance the body's immune response to cancer cells.

[0382]

[0383] Example 3. T1 MRI contrast agent based on polysaccharide cross-linked colloidal particles of the present invention

[0384] In this example, a study was conducted to cross-link various polysaccharides including Dextran (Fig. 8) to form polysaccharide cross-linked colloidal particles and to determine whether they can be used as T1 MRI contrast agents.

[0385] 3-1. Formation of nanostructures using dextran T-5

[0386]

[0387] 180 μmol of dextran T-5 (Fig. 8 (ai), average molecular weight 5,000 Da) 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 h and purified through a 5 kD molecular weight cutoff (MWCO) filter.

[0388] 3-2. Synthesis of nanostructures using maltodextrin

[0389] 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.

[0390] 3-3. Synthesis of nanostructures using alpha-cyclodextrin

[0391] All experiments were performed in the same manner as in Example 3-1, except that alpha-cyclodextrin (Fig. 8 (bi), average molecular weight 970 Da) was used instead of dextran T-5.

[0392] 3-4. Synthesis of nanostructures using beta-cyclodextrin

[0393] All experiments were performed in the same manner as in Example 3-1, except that beta-cyclodextrin (Fig. 8(b-ii), average molecular weight 1100 Da) was used instead of dextran T-5.

[0394] 3-5. Synthesis of nanostructures using inulin

[0395] 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.

[0396] In order to confirm the crosslinking of the materials of Examples 3-1 to 3-5, they were prepared at the same concentration and placed in a cuvette, and the absorbance of the materials was confirmed at a wavelength of 200 to 400 nm using a UV-Vis Spectrophotometer. While general polymers do not show absorbance in the visible light range, when nanoparticles are formed after crosslinking, the transmitted light is scattered, and as a result, absorption is observed to occur in the short wavelength range. Before crosslinking, no absorption was observed in the observation range for the polymer, but for the materials with formed nanostructures, absorption was observed in the short wavelength range (200 to 250 nm), confirming the formation of nanostructures by crosslinking.

[0397] The nanostructures synthesized by Examples 3-1 to 3-5 have amine groups formed on the surface by diethylenetriamine. In order to quantify the number of amine groups on the surface per unit nanostructure, analysis was performed using o-phthalaldehyde assay. As a result, dextran T-5 had 12.7, maltodextrin 4.5, alpha-cyclodextrin 8.1, beta-cyclodextrin 9.0, and inulin 8.0. Therefore, it was confirmed that amine groups were introduced to the surface of all nanostructures using this synthetic method.

[0398] 3-6. Introduction of carboxyl groups into nanostructures

[0399] 25 mg of succinic anhydride was added to the materials of Examples 3-1 to 3-5 at room temperature, and after 24 hours of succinylation reaction, the materials were purified using a 5 kD molecular weight cutoff (MWCO) filter.

[0400] The number of residual amine groups of the materials of Example 3-6 was confirmed through o-phthalaldehyde assay. The number of residual amine groups per unit nanostructure was confirmed 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. If the residual amine is 1 or less, it means that all amine groups have been replaced with carboxyl groups. Therefore, all amine groups of the nanostructures using various polymers in the reaction were replaced with carboxyl groups.

[0401] 3-7. Iron introduction into nanostructures

[0402] 45 uL of ferric chloride hexahydrate solution was added to the material of Example 3-6. After adjusting the pH to 8 with 2.5 M NaOH, the mixture was reacted at room temperature for 1 hour, and then purified using a 5 kD molecular weight cutoff (MWCO) filter to synthesize nanostructures.

[0403] Example 3-7 The iron content and polymer content of the materials were analyzed. The iron bound to the surface 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. As a result, the amount of iron bound to 1 mg of polymer was analyzed 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, respectively. Through this reaction, it was confirmed that similar amounts of iron were introduced during the iron introduction reaction using various polymers.

[0404] 3-8. Measurement of the hydration diameter and surface charge of iron-introduced nanostructures

[0405] Example 3-7 The hydrodynamic diameters of the materials were analyzed using DLS. As a result, it was confirmed that dextran T-5 had a hydrodynamic diameter of 3.6 nm, maltodextrin had a hydrodynamic diameter of 6.8 nm, alpha-cyclodextrin had a hydrodynamic diameter of 2.9 nm, beta-cyclodextrin had a hydrodynamic diameter of 2.8 nm, and inulin had a hydrodynamic diameter of 3.8 nm, which were similar. In terms of surface charge, dextran T-5 had a hydrodynamic diameter of -3.01 mV, maltodextrin had a hydrodynamic diameter of -6.62 mV, alpha-cyclodextrin had a hydrodynamic diameter of -9.06 mV, beta-cyclodextrin had a hydrodynamic diameter of -7.32 mV, and inulin had a hydrodynamic diameter of -2.83 mV. Therefore, it was confirmed that the hydrodynamic diameters and surface charges of the iron-introduced nanostructures formed using this synthetic method were similar.

[0406] 3-9. Comparison of viscosity measurements of nanostructures

[0407] Example 3-7 After the material was prepared at a concentration of 5 mg / mL, the viscosity was measured at 25°C. The viscosities of the cross-linked polymers were similar: dextran T-5 had 1.09 mPa.s, maltodextrin had 1.43 mPa.s, alpha-cyclodextrin had 1.26 mPa.s, beta-cyclodextrin had 1.3 mPa.s, and inulin had 1.14 mPa.s.

[0408] 3-10. Comparison of T1 MRI contrast effects of nanostructures

[0409] In order to analyze the T1 MRI performance of the material in Example 3-7, the spin-spin relaxivity coefficient (r2) and the spin-lattice relaxivity coefficient (r1) were measured, respectively, and their ratio (r2 / r1ratio) was calculated. The r2 / r1ratio is a measure to determine whether a contrast agent is suitable as a T1 MRI contrast agent or a T2 MRI contrast agent. As a result of analyzing the material in Example 3-7 at 3.0 Tesla MRI, dextran had an r11.90, r22.27, r2 / r1 ratio 1.19, maltodextrin is r14.60, r25.19, r2 / r1 ratio 1.13, alpha-cyclodextrin has r15.26, r25.78, r2 / r1 ratio 1.10, beta-cyclodextrin has r15.62, r26.24, r2 / r1 ratio 1.11, inulin is r14.33, r24.73, The r2 / r1 ratio was confirmed to be 1.09. Therefore, it was confirmed that all synthesized substances had an r2 / r1 ratio close to 1 and thus exhibited T1 MRI contrast effects.

[0410] 3-11. Evaluation of the animal imaging efficacy of iron-introduced nanostructures

[0411] After intravenous administration of the material in Example 3-7 to mice, T1-weighted images were acquired using a 9.4 Tesla MRI. Male Balb / c mice, 5 weeks or older, were anesthetized, and the material in Example 3-7 was administered into the tail vein. T1 MRI images were then acquired hourly and the signal-to-noise ratio (SNR) was analyzed. As illustrated in Fig. 9, all iron-incorporated nanostructures of Example 3-7 exhibited bright signals in blood vessels (jugular veins) approximately 3 to 5 minutes after injection.

[0412] 3-12. Elongation of iron-introduced nanostructures

[0413] In Example 3-7, after intravenous administration of the substance to animals, T1-weighted images were acquired for up to 1 hour using a 9.4 Tesla MRI to confirm renal excretion of the administered substance. As shown in Figure 10, most iron-incorporated nanostructures began to exhibit T1 signals in the bladder within 30 minutes. This indicates that all administered substances passed through the kidneys and were excreted into the bladder.

[0414]

[0415] Example 4. MR arthrography using NEMO-103, a T1 MRI contrast agent based on dextran crosslinker.

[0416] 4-1. Preparation of NEMO-103, a T1 MRI contrast agent based on dextran crosslinker

[0417] After dissolving dextran T-10 (20 mM) in an aqueous solution, sodium hydroxide and epichlorohydrin (ECH) were sequentially added, followed by the addition of ethylenediamine (EDA) to induce crosslinking of dextran molecules (10,000 g / mol). At this time, EDA molecules that did not participate in crosslinking provided amine terminal groups on the core surface, and the amine groups were substituted with carboxyl groups by reacting with succinic anhydride.

[0418] The dextran crosslinker thus manufactured 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 on the carboxyl groups of the core surface. This was further purified using a 10 kDa MWCO filter, and finally, a water-soluble dextran crosslinker-based T1 contrast agent, NEMO-103 injection, was obtained.

[0419] NEMO-103 is an amorphous colloidal nanoparticle composed of iron (Fe) bound to a dextran crosslinker core. The average hydrodynamic diameter of the manufactured 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 -OH functional groups were modified by the crosslinker, and this high degree of crosslinking prevented particle aggregation and imparted structural stability by forming a high-density three-dimensional network.

[0420] The contrast agent remained stable without aggregation for more than 11 days under normal physiological conditions as well as at various pH (5, 7, 9) and salt concentrations (250, 500, 1,000 mM NaCl), which meets the stability criteria that are much longer than the elimination period of 24-48 hours confirmed in nonclinical studies.

[0421] As a result of the magnetic property analysis, the magnetization value in a 3 T magnetic field based on the MPMS (Magnetic Property Measurement System) was measured to be 6.53 emu / gFe, and the r2 / r1 ratio was 1.3 as a result of measuring the T1 and T2 relaxation coefficients according to the iron concentration in the MRI phantom experiment, which indicates ideal relaxivity characteristics as a T1 contrast agent.

[0422] 4-2. Nonclinical imaging performance and immunological safety evaluation of NEMO-103, a T1 contrast agent based on dextran crosslinker.

[0423] Contrast agent NEMO-103 (same name: INV-002) is a T1 MRI contrast agent based on water-soluble polysaccharide cross-linked colloidal particles having a compact spherical three-dimensional network structure formed by selectively modifying the monosaccharide -OH functional groups of dextran polymers with an epoxide-based first cross-linking agent, followed by intra- or intermolecular reaction with a second cross-linking agent (e.g., containing a polyvalent amine group).

[0424] This structure is designed to maximize immune evasion properties by suppressing nonspecific binding to recognition receptors (BCRs, PRRs, etc.) on the surface of immune cells, as well as ensuring physicochemical stability. This example presents nonclinical data, including safety assessments related to the potential for inducing an immune response, as well as imaging performance.

[0425] 4-2-1. Nonclinical contrast performance evaluation (CNR-based validity analysis)

[0426] The image enhancement effect of NEMO-103 was evaluated based on the contrast-to-noise ratio (CNR) in the following areas:

[0427] Assessment sites: meniscus, anterior cruciate ligament, cartilage, bone

[0428] Average CNR improvement: More than 120% improvement compared to existing Gd-based contrast agents.

[0429] Contrast duration: increased by at least 4 times

[0430] These results suggest that NEMO-103 can obtain high-contrast images of tissues, providing a basis for improving image resolution and accuracy of joint diagnosis.

[0431] 4-2-2. Toxicity Assessment Based on Immunosafety (GLP Toxicity Testing and Immunological Interpretation)

[0432] NEMO-103 met the preclinical safety requirements according to US FDA guidelines, and the following characteristics indirectly suggest that it is a non-immunogenic particle:

[0433] HEK-293 cell toxicity test: IC 50 No extraction, no cytotoxicity

[0434] hERG assay: no cardiac repolarization impairment

[0435] Platelet / plasma mixture test: no coagulation reaction

[0436] Plasma protein binding: Low levels were measured, indicating low potential for tissue accumulation.

[0437] In particular, the cross-linked structure of NEMO-103 was designed based on selective modification of the monosaccharide -OH functional group and precisely controlled cross-linking conditions, which indirectly suggests that it is a non-immunogenic particle at the molecular level as follows:

[0438] Avoiding binding to PRRs and BCRs: Reduced binding to receptors that recognize polysaccharide structural specificity.

[0439] Clustering inhibition: Does not act as a multivalent antigen and thus does not induce immune signaling

[0440] No immune cell activation: No B cell activation or antibody production even with repeated administration

[0441] Stability under pathological conditions: No hypersensitivity reactions even in inflammatory lesions

[0442] 4-2-3. Summary of key toxicity test data (including exposure multiples compared to the clinically anticipated dose)

[0443]

[0444] Additionally, it was judged to be negative in all of the reverse mutation tests, chromosome aberration tests, and micronucleus tests, and there were no concerns about carcinogenicity, genotoxicity, or reproductive toxicity.

[0445] Example 4-2 indirectly demonstrated that NEMO-103, a T1 contrast agent based on non-immunogenic polysaccharide cross-linked colloidal particles, is a next-generation MRI contrast agent that boasts enhanced contrast performance and immunological safety compared to existing Gd contrast agents. In particular, the absence of immune responses even after repeated administration and the absence of toxicity observed even at high doses suggest that the platform technology of the present invention has broad applicability in various fields of bioimaging and precision diagnosis.

[0446] 4-3. Comparison of image quality in shoulder MR arthrography

[0447] To evaluate the clinical efficacy of NEMO-103, image quality was compared after intra-articular administration of NEMO-103 to the shoulder joint in 32 patients (total of 80 MRA images) in a phase 1 / 2a trial.

[0448] Comparison 1: Quality comparison between Phase I (30 minutes) and Phase II (60 minutes) images in the same patient showed no significant differences in CNR, joint capsule distensibility, and overall image quality (Fig. 11).

[0449] Comparison 2: In the comparison of NEMO-103 and GBCA-based images at the Phase I time point, the CNR was similar, but the degree of distension of the inferior joint capsule and axillary pouch was significantly higher in NEMO-103, and the overall image quality evaluation also showed statistically superior results (p < 0.05, Fig. 12).

[0450] Comparison 3: The difference was more pronounced at the Phase II time point (approximately 54-55 minutes), with NEMO-103-based images being significantly superior to GBCA in CNR, inferior capsular distension, and axillary pouch distension (p < 0.01, Fig. 13).

[0451] 4-4. Visual Turing Test (VTT)

[0452] The discriminability of images acquired at two different time points (Phase I and II) after NEMO-103 injection was evaluated by radiologists. The average discriminatory accuracy among eight readers was 46.8% (146 / 312), which was not statistically significantly different from random guessing (p = 0.423).

[0453] 4-5. Safety Evaluation in Phase 1 / 2a Clinical Trials

[0454] The safety of NEMO-103 was evaluated in a clinical trial (Stage 1: n=9, Stage 2: n=23) involving 32 patients with suspected rotator cuff lesions due to shoulder pain.

[0455] 4-5-1. Adverse reactions:

[0456] Two adverse reactions (ADRs) occurred, but both recovered. There were no serious adverse reactions (SAEs) or drug-related withdrawals. No dose-limiting toxicities (DLTs) were observed.

[0457] 4-5-2. Persistence and Tissue Safety:

[0458] Tissue retention of NEMO-103 in the liver and spleen was confirmed to be negative (100% disappearance) 24 hours after administration, as confirmed by imaging studies. The signal difference between pre- and post-administration liver and spleen MRI was positive (liver: 13.2 ± 4.0, spleen: 5.5 ± 0.9).

[0459] 4-5-3. Video Quality and Sustainability:

[0460] Similar image quality was achieved in images acquired 30 and 60 minutes after administration, and excellent performance was also demonstrated in depicting radiological structures (joint capsule, rotator cuff, cartilage, etc.). Notably, NEMO-103, with its long retention time, maintained excellent CNR even after 2 hours and was completely eliminated from the joint space after 24 hours, eliminating the possibility of body retention.

[0461] 4-6. Evaluation of the Correlation Between Joint Distension and MRA Image Quality in a Phase 2b Clinical Trial

[0462] This example is based on the results of a phase 2b clinical trial conducted to evaluate the intra-articular distribution and contrast efficacy of NEMO-103, a dextran-crosslinked T1 MRI contrast agent. The trial set joint distension, joint boundary sharpness, and image contrast as the main evaluation items of image quality, and confirmed statistical significance (p-value < 0.0001) in both the primary and secondary analysis groups (see Fig. 14).

[0463] As described below, this example demonstrated the efficacy and safety of NEMO-103 as a T1 contrast agent for achieving high-resolution intra-articular MRA imaging in clinical practice, and in particular, demonstrated the possibility of AI-based quantitative image analysis based on the correlation between joint swelling and image quality.

[0464] 1. Intra-articular contrast agent distribution and diagnostic value

[0465] Joint swelling is an imaging index that quantifies the uniformity of distribution and extent of penetration of T1 contrast agent. It enhances the visualization of intra-articular anatomical structures (cartilage, synovium, ligaments, etc.) and increases diagnostic sensitivity and specificity. In this study, enhanced joint swelling was closely associated with improved image sharpness and contrast, indirectly reflecting the in vivo uniformity of contrast agent distribution and prolonged residence time.

[0466] 2. Clinical evidence for immunodeactivation properties

[0467] NEMO-103 is designed as a non-immunogenic polysaccharide cross-linked colloidal particle-based contrast agent, and its characteristics were supported in this trial by the following immunological safety indicators:

[0468] Absence of acute immune response: No immune response-based abnormalities such as synovitis, swelling, or pain were observed after intra-articular administration.

[0469] Maintaining normal joint swelling: A stable increase in swelling without excessive swelling or intra-articular reaction indirectly suggests the absence of immune hypersensitivity.

[0470] Uniformity of contrast agent distribution and image stability: Together with the physicochemical properties of the contrast agent, stable image quality can be secured even in the inflammatory environment of the body.

[0471] 3. AI-based image analysis and standardization potential

[0472] Quantitative imaging metrics such as joint swelling, sharpness, and contrast can serve 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 study serve as repeatable criteria for image quality, enhancing the reliability of contrast agent evaluation.

[0473]

[0474] Example 5. Magnetic resonance lymphangiography using INV-001

[0475] 5-1. Preparation and Characterization of Contrast Agents

[0476] Gd-DOTA (control):

[0477] DOTAREM® (Guerbet, France) was used after being diluted with saline (final concentration: 15 mM).

[0478] INV-001 (test group):

[0479] Dextran T-5 (20 mM) with an average molecular weight of 5,000 Da was reacted with sodium hydroxide and epichlorohydrin, followed by the addition of diethylenetriamine to produce cross-linked dextran. The amine terminus was then modified with carboxyl groups using succinic anhydride, and iron (III) chloride was reacted to coordinate the iron to the surface. The product was purified and concentrated using a 3 kDa MWCO filter.

[0480] Structural characteristics:

[0481] TEM analysis revealed that INV-001 was a spherical particle with an average diameter of 2.6 ± 0.3 nm (n = 100), and negative staining was performed with uranyl acetate. Dynamic light scattering analysis revealed that the hydrodynamic size was 3.6 ± 0.2 nm, and the surface charge was measured to be -2.95 ± 0.20 mV. It was confirmed that more than 60% of the total number of monosaccharides constituting INV-001 was modified by the cross-linking agent, and an average of 5 iron atoms were present per particle.

[0482] Physicochemical properties:

[0483] Magnetization: 14.25 emu / gFe (3 Tesla magnetic field)

[0484] Relaxivity: r₁ = 3.95 ± 0.35 mM¹s¹, r₂ = 4.73 ± 0.24 mM¹s¹, r₂ / r₁ = 1.20 ± 0.04

[0485] Other: pH = 8.23, viscosity = 3.23 ± 0.03 cP, density = 1.031 ± 0.001 g / cm³, boiling point = 101°C, freezing point = 0°C

[0486] Stability Assessment:

[0487] INV-001 maintained a stable colloidal state without aggregation even under pH 5, 7, and 9 conditions and 250–1,000 mM NaCl, and there was no change in particle size during the period from in vivo administration to excretion.

[0488] 5-2. Fluid dynamic size and magnetic resonance relaxivity

[0489] The hydrodynamic size of INV-001 was measured to be 3.6 nm, and the T₁ 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. As a result, r₁ = 2.61 mM¹s¹, r₂ = 4.33 mM¹s¹, and r₂ / r₁ = 1.66.

[0490] This means that INV-001 is suitable as a contrast agent that specifically remains in the lymphatic system without venous contamination, as it exceeds the maximum size (2 nm) that can penetrate into veins.

[0491] 5-3. Video Performance Comparison: INV-001 vs. Gd-DOTA (see Figure 15)

[0492] This study compared the pharmacokinetic properties and imaging performance of the iron-based contrast agent INV-001 and the gadolinium-based contrast agent (Gd-DOTA) in the lymphatic system using magnetic resonance lymphangiography (MRL) in Sprague-Dawley (SD) rats. In particular, INV-001 is a novel contrast agent designed to prevent venous contamination, and this study evaluated the optimal dosage and image quality for visualizing lymph nodes and lymphatic vessels.

[0493] After administering 1.125 μmol (75 μL, 15 mM) of INV-001 and Gd-DOTA, respectively, the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of lymph nodes and lymphatic vessels were compared.

[0494]

[0495] While Gd-DOTA showed a signal peak at 16 minutes after administration, INV-001 maintained a uniform high signal for a longer duration (16–32 minutes) and showed excellent contrast enhancement, especially in lymphatic vessels (CNRLV).

[0496] In this way, INV-001 was confirmed to have the promise of being an iron-based MRL contrast agent that has less venous contamination than Gd-based contrast agents and can provide a long-term stable contrast effect in lymph nodes and lymph vessels.

[0497] 5-4. Qualitative comparison of venous contamination, skin reflux, and lymph node congestion.

[0498] In this example, after subcutaneous injection of INV-001 or Gd-DOTA into the lower extremities of Sprague-Dawley rats, 3D TOF-based magnetic resonance lymphangiography (MRL) was performed to qualitatively evaluate the presence or absence of venous contamination, dermal backflow, and lymph node congestion. The evaluation was performed according to the qualitative evaluation criteria in Table 6.

[0499]

[0500] In the Gd-DOTA-administered groups, venous contamination was observed in both the 1.125 μmol and 2.25 μmol groups, with the 2.25 μmol group showing the most severe findings. In contrast, INV-001 successfully visualized lymph nodes and lymphatic vessels without venous contamination under all administration conditions. Furthermore, tissue confirmation using methylene blue 2 days after MRL imaging confirmed that the lymphatic structures observed in the INV-001-based images were consistent with the anatomical entity.

[0501] 5-5. Qualitative image quality assessment according to various injection conditions

[0502] To comprehensively evaluate venous contamination and image quality, MRL images of the popliteal lymph nodes and lymphatic vessels were acquired after subcutaneous injection of INV-001 and Gd-DOTA under various conditions, and qualitative scoring was performed according to Table 6.

[0503] As a result of changing the concentration under the condition of fixed injection volume (75 μL), INV-001 obtained excellent scores of 4.2, 5.0, and 5.0 in the 0.75 μmol (10 mM), 1.125 μmol (15 mM), and 1.5 μmol (20 mM) administration groups, respectively. On the other hand, the 2.25 μmol (30 mM) Gd-DOTA administration group showed a low score of 3.8.

[0504] As a result of changing the injection amount under fixed concentration (15 mM) conditions, INV-001 showed scores of 3.6, 4.4, and 4.6 in the 0.3 μmol (20 μL), 0.45 μmol (30 μL), and 0.75 μmol (50 μL) administration groups, respectively. Gd-DOTA 1.125 μmol (75 μL) only scored 3.2.

[0505] As confirmed in Figure 16, INV-001-based MRL images showed no venous contamination or skin reflux compared to Gd-DOTA, and the image contrast of lymph nodes and lymphatic vessels was also excellent. In particular, the highest level of image quality was confirmed under the 0.45 μmol and 0.75 μmol conditions.

[0506] This Example 5-5 experimentally demonstrated that the iron-based contrast agent INV-001 has superior biocompatibility and lymphatic vessel-specific imaging properties compared to Gd-DOTA. In particular, INV-001 has a hydrodynamic diameter of approximately 3.6 nm, which exceeds the venous penetration limit (approximately 2 nm), fundamentally preventing venous contamination, and exhibited a long visualization duration and excellent contrast effect in lymph nodes. As a result of the biosafety evaluation, no residual INV-001 was observed in the tissues within 24 and 48 hours after administration, and no changes in liver and kidney signals were observed, confirming rapid elimination from the body.

[0507] Therefore, INV-001 has high potential as a T1 contrast agent for magnetic resonance lymphangiography (MRL) and is a promising candidate that can provide long-term stable image quality without venous contamination in the diagnosis and monitoring of lymphatic diseases.

[0508] 5-6. Evaluation of the Lymphangiographic Efficacy of INV-001 in Various Animal Models

[0509] In this example, the lymphatic vessel-specific imaging ability of INV-001 contrast agent was evaluated in various animal models (Sprague-Dawley rats, miniature pigs, and beagle dogs) and compared with that of a gadolinium-based contrast agent (Gd-DOTA). Each experiment was evaluated with a focus on the degree of lymphatic vessel visualization and the presence of venous contamination, and the image analysis results are depicted in Figure 17.

[0510] 1) Sprague-Dawley rat (MRL image)

[0511] After subcutaneous injection of INV-001 or Gd-DOTA into the hind limbs of SD rats, 3D TOF-based MRL imaging was performed. In the INV-001-injected group, peripheral lymphatic vessels with diameters of 300 μm or less were clearly visualized, and no venous contamination was observed. In contrast, when Gd-DOTA was administered under the same conditions, venous contamination was clearly observed.

[0512] 2) Mini pig model

[0513] MRL was performed after injection of INV-001 or Gd-DOTA under conditions corresponding to the low dose of the clinical trial (15 mM, 0.6 mL × 3 sites). When INV-001 was administered, abdominal lymph nodes and leg lymphatic vessels were clearly visualized, and venous contamination was not observed. In contrast, when Gd-DOTA was administered, lymphatic branching appeared faintly, and venous structures were enhanced, clearly showing contamination.

[0514] 3) Beagle dog model

[0515] After INV-001 was injected into the dorsum of the foot, lymphatic vessels were clearly visualized. Notably, lymphatic vessel structures were observed only in the INV-001-treated group, compared to the untreated control group. This suggests that the contrast effect of INV-001 was effectively delivered to the skin lymphatic vessels and intramuscular lymphatic vessels.

[0516] 4) Validation of efficacy in disease models

[0517] In a lymphadenectomy disease model using minipigs, the effects of INV-001 injection on lymphangiography were compared between the left lymphadenectomy group and the normal right group. Imaging results showed that no lymph nodes were identified on the resected left side, whereas lymphatic branching and lymph nodes were clearly enhanced in the normal right side. This demonstrates that INV-001 can sensitively reflect changes in lymphatic structure even in disease states.

[0518] 5-7. Contrast enhancement observed in the central lymphatic system after peripheral administration of INV-001 in beagle dogs.

[0519] This example evaluated whether INV-001, an iron-based T1 MRI contrast agent, was injected subcutaneously into the lower extremities of a beagle dog and then magnetic resonance lymphangiography (MRL) was used to assess whether the contrast agent reached the central lymphatic system as well as the peripheral lymphatic system.

[0520] As illustrated in Figure 18, INV-001 exhibited extensive contrast enhancement, beginning at the injection site in the distal lower extremity and ascending along the popliteal lymph nodes and peripheral lymphatic vessels, to the central lymphatic region of the abdomen. Notably, diffusion into the central lymphatic system was observed without signal loss in the lymph nodes, suggesting that the contrast agent can continuously travel along the lymphatic flow without being captured by immune cells in the lymph nodes.

[0521] These results indirectly demonstrate that the contrast agent component of INV-001 significantly inhibits the interaction with immune cells in the body, especially immune cells in lymph nodes such as B cells and dendritic cells. This is due to the structural characteristics of the non-immunogenic polysaccharide cross-linked colloidal particles of the present invention. Specifically, (a-1) the -OH functional group of the monosaccharide is selectively modified with a first cross-linking agent of the epoxide series, (a-2) a three-dimensional network is formed between the modified -OH functional groups through a second cross-linking agent (e.g., polyvalent amine) or direct reaction, and (a-3) the surface charge is adjusted to the range of -20 mV to 0 mV by further modifying the amine functional group derived from the cross-linking agent with a carboxylic acid group, thereby reducing the binding affinity to the PRR or BCR of the immune cell.

[0522] As a result, INV-001 (i) minimizes receptor-ligand interactions with numerous immune cells in lymph nodes, preventing signal transduction through receptor clustering; (ii) does not induce B cell activation or antibody production; (iii) maintains resistance to hydrolysis by body enzymes; and (iv) enables stable excretion without hypersensitivity immune responses even under inflammatory conditions.

[0523] These technical characteristics are demonstrated through the imaging experiment in Fig. 18 that INV-001 can penetrate the lymph node structure densely populated with immune cells and reach the central lymphatic system along the flow of lymph fluid, which strongly supports the applicability of the contrast agent of the present invention as a non-immunogenic T1 lymphatic contrast agent platform.

[0524] 5-8. GLP Nonclinical Toxicity Evaluation and Immune Evasion Design Validation of INV-001, a Non-Immunogenic Polysaccharide Cross-Linked Colloidal Contrast Agent

[0525] This example summarizes the results of a non-clinical toxicity test conducted under GLP standards to confirm that INV-001, an iron-based T1 contrast agent of the present invention, exhibits excellent biosafety under high-dose and repeated administration conditions without inducing an immune response in the body.

[0526] Conventional polysaccharide-based drugs or nanoparticles can interact with immune cells (e.g., B cell receptors, pattern recognition receptors), potentially triggering unexpected immune responses (e.g., antibody production, inflammation, hypersensitivity, allergies). In particular, cross-linked polysaccharide-based contrast agents can accumulate in the body and persist for long periods due to their polymeric structure, raising concerns about immunotoxicity and reduced contrast agent efficacy.

[0527] INV-001 of the present invention is designed as a non-immunogenic polysaccharide cross-linked colloidal particle platform to avoid B cell activation and antibody production, inhibit receptor clustering, and enable stable excretion without long-term retention.

[0528] As shown in the table below, INV-001 did not show any toxic reactions to the cardiovascular, central nervous, respiratory, immune, and major organ systems even at doses several tens of times higher than the clinically planned dose, and was confirmed to be completely excreted from the body without accumulation.

[0529]

[0530] Additionally, the possibility of genotoxicity was ruled out as it showed negative results in the reverse mutation test, chromosome aberration test, and micronucleus test.

[0531] To address the immunological and pharmacodynamic limitations of existing gadolinium-based contrast agents, INV-001 of the present invention utilizes a molecular design strategy that blocks nonspecific binding to immune cell receptors. This contrast agent demonstrated immune evasion and reduced residual activity, as evidenced by imaging results (see Figure 18 and Examples 5-7) demonstrating stable transport to the central lymphatic system without being phagocytosed or retained by immune cells after passing through lymph nodes.

[0532] In addition, it was proven through GLP test results that it is a non-immunogenic contrast agent that is safely excreted without antibody production or tissue reaction even when administered repeatedly, and significantly reduces the possibility of immune-based side effects (allergy, inflammation, etc.) that accompany polysaccharide-based drugs.

[0533] Through this example, INV-001 was confirmed to be a highly safe contrast agent that does not accumulate in vivo and is excreted. Furthermore, its structural characteristics, which weaken its binding affinity to immune cell receptors, have been shown to suppress immune activation upon repeated administration. Therefore, INV-001 can be utilized as a lymphatic-specific T1 contrast agent with a low risk of inducing an immune response and guaranteed safety even under high-dose and repeated administration conditions.

[0534]

[0535] Example 6. Evaluation of Lymphatic System-Specific Imaging Efficacy and Immune Inactivation-Based Safety in Phase 1 Clinical Trial of INV-001

[0536] This example relates to the results of a phase 1 clinical trial to evaluate the image quality and safety of INV-001 when applied to humans, particularly its responsiveness to the immune system.

[0537] After intradermal or subcutaneous injection of INV-001 into the distal foot, high-resolution magnetic resonance lymphangiography (MRL) was performed. Results showed that INV-001, originating from the injection site, migrated along the peripheral lymphatic vessels, resulting in clear and distinct lymphatic vessel images (see Figure 19). The images reflected the distribution of contrast agent within the lymphatic system and enabled lymphatic-specific, high-resolution visualization over time after injection.

[0538] 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), administered to a total of nine healthy adult volunteers (see Table 8).

[0539]

[0540] As a result, the following key indicators of immune safety were identified:

[0541] Maximum Tolerated Dose (MTD) not reached: No maximum tolerated dose (MTD) due to toxicity was observed in any dose group, suggesting that no significant immune-related adverse reactions, such as systemic inflammation, febrile reaction, or cytokine storm, occurred even at high doses.

[0542] 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 subjects. This means that no immune-related adverse events, such as allergies, rashes, or vasculitis, were clinically identified.

[0543] Successful Lymphatic System-Specific Imaging: Despite the fact that lymph nodes and lymphatic vessels are densely populated with immune cells, INV-001 reliably imaged lymph nodes, and no image degradation or local inflammatory response due to interaction with immune cells was observed. This indirectly demonstrates that INV-001 was not recognized as an immunogenic substance by T cells, B cells, or macrophages in the lymph nodes.

[0544] In particular, INV-001 is a water-soluble polysaccharide cross-linked colloidal particle with a compact, spherical three-dimensional network structure formed by selectively modifying the monosaccharide -OH functional group and precisely controlling the cross-linking agent conditions. It exhibits significantly low binding affinity to immune cell receptors such as the B cell receptor (BCR) and PRR, resulting in a very low likelihood of inducing an immune response. This structural characteristic is directly related to the non-immunogenicity confirmed in the phase 1 clinical trial results.

[0545] These results suggest that INV-001 demonstrates both safety and efficacy as a T1 MRI contrast agent capable of precisely visualizing the lymphatic system without immune-related adverse reactions in humans, supporting its potential for future clinical use in the diagnosis and monitoring of lymphatic diseases.

[0546]

[0547] Example 7. INV-MRL-based contrast technique for staging lymphedema: Application of non-immunogenic polysaccharide cross-linked colloidal particles.

[0548] This example describes the results of applying magnetic resonance lymphangiography (INV-MRL) using INV-001, a contrast agent based on chemically cross-linked, non-immunogenic polysaccharide cross-linked colloidal particles, to effectively visualize lymphatic lesions in an animal model of lymphedema and to assess the stage of the disease.

[0549] The contrast agent used in this example is a colloidal particle with a highly precise three-dimensional network structure formed by selectively modifying the -OH functional group of a monosaccharide with an epoxide-based first cross-linking agent, followed by a second cross-linking agent (including a polyvalent amine) or direct cross-linking reaction between adjacent -OH functional groups. This particle is designed to avoid interaction with in vivo immune cells (e.g., BCR, PRR, etc.) by controlling the surface charge within the range of -20 mV to 0 mV by replacing some or all of the amine groups derived from the cross-linking agent with -COOH functional groups. This structural design prevents clustering of immune cell receptors and imparts non-immunogenic properties that do not induce immune activation even upon repeated administration.

[0550] Figure 20 is a representative image showing the results of applying INV-MRL and NIRF-ICGL, respectively, in an animal model induced with lymphedema. INV-MRL clearly visualizes deep lymphatic vessels even in areas where contrast agent diffuses along the skin surface, suggesting its advantage in detecting structural abnormalities that are difficult to detect with NIRF-ICGL. In particular, as indicated by the orange arrow in Figure 20, INV-MRL was able to visualize hidden collateral lymphatic vessels (collateral LV) beneath the diffused contrast agent.

[0551] INV-MRL could also qualitatively identify structural abnormalities such as lymphatic leakage, collateral channel formation, and lymphatic dilatation according to the stage of lymphedema classified from 0 to 4, and these abnormalities showed a high correlation with the splash, stardust, and diffusion patterns defined by NIRF-ICGL. The thresholded area ratio (TAR) value derived from the same ROI showed a quantitative correlation that increased with the progression of the stage, and the TAR value measured in INV-MRL showed higher sensitivity than NIRF-ICGL (R²=0.5919 vs. 0.5309, dorsal NIRF).

[0552] In particular, INV-MRL offers the following technical advantages:

[0553] Based on non-immunogenic polysaccharide cross-linked colloidal particles, retention in immune organs such as lymph nodes is limited.

[0554] Unlike GBCA-based MRL, it selectively visualizes only lymph flow without venous contamination.

[0555] Low dependence on ROI and reproducibility of deep lymphatic structures under the same conditions.

[0556] 3D reconstruction capabilities enable quantitative analysis of the extent of lymphatic diffusion as the disease progresses.

[0557] Through these technical validations, INV-MRL is evaluated as an excellent imaging platform for visualizing deep lymphatic structures compared to conventional optical imaging-based NIRF-ICGL, and the applicability of non-immunogenic polysaccharide cross-linked colloidal particles to the field of lymphatic diagnosis is demonstrated.

[0558] This example demonstrates at a preclinical level that the design logic and structural characteristics of non-immunogenic polysaccharide cross-linked particles, which are the main technical components of the present invention, can provide a new paradigm for lymphatic imaging technology, and provides a technological foundation that can establish a new standard for high-precision lymphatic imaging and staging assessment in future clinical applications.

Claims

1. A water-soluble polysaccharide cross-linked colloid particle in which the -OH functional group of the cross-linking target polysaccharide dispersed in an aqueous solvent is modified with a cross-linking agent and the surface charge of the particle is controlled within the range of -20 mV to 0 mV through the -COOH functional group exposed on the surface, thereby reducing or minimizing immunogenicity to immune cells compared to the cross-linking target polysaccharide and being excreted from the body without being hydrolyzed by enzymes in the body. A polysaccharide cross-linked colloidal particle is a non-immunogenic polysaccharide colloidal particle characterized in that (i) the -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-linking agent having an epoxide group in an aqueous solvent, (a) the functional group modified with the first cross-linking agent and a spatially adjacent -OH functional group are cross-linked intramolecularly and / or intermolecularly via a second cross-linking agent having two or more amine groups (-NH2) to form the polysaccharide cross-linked particle, and (ii) the number of basic amine groups derived from the cross-linking agent exposed on the surface is controlled by modification with a -COOH functional group.

2. A non-immunogenic polysaccharide cross-linked colloidal particle characterized in that, in paragraph 1, 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are building blocks of the polysaccharide, are modified by a cross-linking agent so that they are not hydrolyzed by enzymes in the body and immunogenicity against immune cells is reduced or minimized.

3. In the first paragraph, the polysaccharide cross-linked colloidal particle is a non-immunogenic polysaccharide cross-linked colloidal particle characterized in that at least one -OH functional group of at least one monosaccharide among two consecutive monosaccharides in the polysaccharide chain is mostly modified with a cross-linking agent, thereby not being hydrolyzed by enzymes in the body and having reduced or minimized immunogenicity to immune cells.

4. In the first paragraph, the polysaccharide cross-linked colloidal particle is characterized by a non-immunogenic polysaccharide cross-linked colloidal particle that (a) is filtered in the kidney but does not penetrate the blood vessel walls of normal capillaries and (b) is optionally excreted only into the distal lymphatic vessels, and has a hydrated diameter of 2 to 10 nm, preferably 8 nm or less, more preferably 6 nm or less.

5. Non-immunogenic polysaccharide cross-linked colloidal particles according to claim 4, characterized in that after administration to the body, the polysaccharide cross-linked colloidal particles enter the blood vessels of the nephron and are excreted in urine through the filtration mechanism of the kidney.

6. In the fourth paragraph, the polysaccharide cross-linked colloidal particles are non-immunogenic polysaccharide cross-linked colloidal particles that are selectively discharged only into the distal lymphatic vessels without penetrating or being discharged into the capillaries of the administration site, and are characterized in that an amount corresponding to at least 50% of the injected amount remains at the administration site for at least 1 hour, preferably at least 2 hours, before being discharged through the lymphatic vessels.

7. A non-immunogenic polysaccharide cross-linked colloidal particle characterized by extending the residence time at the injection site in accordance with paragraph 6, thereby inducing interstitial space enhancement and / or anatomical space distension at the injection site.

8. A non-immunogenic polysaccharide cross-linked colloidal particle, characterized in that the polysaccharide cross-linked colloidal particle itself is not uptaken by cells unless it is targeted with a ligand having affinity for a cell surface receptor in the first paragraph.

9. In the first paragraph, the polysaccharide cross-linked colloidal particle has an iron ion, a gadolinium ion (Gd) on the cross-linking agent-derived amine functional group or -COOH functional group exposed on the surface. 3+ ) or manganese ion (Mn 2+ ) or non-immunogenic polysaccharide cross-linked colloidal particles characterized by their coordination bonding with iron oxide particles and their use as MRI contrast agents.

10. In the first paragraph, a non-immunogenic polysaccharide cross-linked colloidal particle characterized in that the polysaccharide cross-linked colloidal particle to which the metal ion is coordinated simultaneously acts as a chelator of the metal ion through an amine functional group or a -COOH functional group derived from a cross-linking agent to which the metal ion is not coordinated.

11. A non-immunogenic polysaccharide cross-linked colloidal particle according to any one of claims 1 to 10, characterized in that it is provided by a manufacturing method comprising the following steps: The first step is to prepare an aqueous solution of linear polysaccharides, branched polysaccharides, or cyclic polysaccharides; A second step of modifying the -OH functional group of a monosaccharide, which is a building block of a polysaccharide, with the first cross-linking agent by adding dropwise a first cross-linking agent having an epoxide functional group that reacts with an alkaline aqueous solution and the hydroxyl group (-OH) of the polysaccharide; A third step of adding a second cross-linking agent having two or more amine groups (-NH2) dropwise so that spatially adjacent functional groups modified with the first cross-linking agent are cross-linked intramolecularly and / or intermolecularly through the second cross-linking agent, thereby generating polysaccharide cross-linked colloidal particles having terminal amine groups derived from the second cross-linking agent on the surface; A fourth step of modifying some or all of the terminal amine groups of a polysaccharide cross-linked colloidal particle having a terminal amine group derived from a second cross-linking agent on the surface by administering an organic acid anhydride to the particle to form a carboxylic acid group and / or a carboxylate group; and Optionally, the water-dispersible polysaccharide cross-linked colloidal particles prepared in the previous step are added with iron ions (Fe 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+ ) by administering a precursor or aqueous solution of iron oxide nanoparticles, (i) by introducing iron ions (Fe) to the amine functional groups or -COOH functional groups derived from the cross-linker exposed on the surface. 2+ / 3+ ), gadolinium ion (Gd 3+ ) or manganese ion (Mn 2+ ) A fifth step of preparing a complex in which the surface of the polysaccharide-crosslinked colloidal particles or (ii) iron oxide nanoparticles is modified with the polysaccharide-crosslinked colloidal particles.

12. A non-immunogenic polysaccharide cross-linked colloidal particle according to any one of claims 1 to 10, characterized in that the polysaccharide cross-linked colloidal particle does not cause an immune response as an antigen, does not produce antibodies against the antigen, can be administered repeatedly, does not cause cell damage or tissue damage due to an inflammatory response, or does not induce a chronic inflammatory disease.

13. In any one of claims 1 to 10, a non-immunogenic polysaccharide cross-linked colloidal particle characterized in that the immunogenicity for immune cells is reduced or minimized when the polysaccharide cross-linked colloidal particle is distributed in the body and is not recognized as a foreign antigen by the immune system, does not produce antibodies against the polysaccharide cross-linked colloidal particle, does not cause hypersensitivity reactions or immune-mediated adverse effects, does not activate the complement system, does not cause an inflammatory reaction, is not phagocytosed by macrophages upon repeated administration, is not recognized or bound by pattern recognition receptors (PRRs), does not release cytokines, or does not stimulate the NF-κB activation pathway in macrophages.

14. In paragraph 12, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or 10 times the administered dose 2 Non-immunogenic polysaccharide cross-linked colloidal particles characterized by no inflammation or hypersensitivity reactions even at high doses.

15. A non-immunogenic polysaccharide cross-linked colloidal particle according to any one of claims 1 to 10, characterized in that the polysaccharide cross-linked colloidal particle is a non-immunostimulatory particle that is not an immunogen or antigen that binds directly to a cell membrane receptor of an immune cell or indirectly via an antibody to an immune cell or non-immune cell having an Fc receptor.

16. A non-immunogenic polysaccharide cross-linked colloidal particle according to any one of claims 1 to 10, characterized in that the polysaccharide cross-linked colloidal particle does not activate or proliferate B cells or mast cells when distributed in the body.

17. A non-immunogenic polysaccharide cross-linked colloidal particle according to any one of claims 1 to 10, characterized in that the polysaccharide cross-linked colloidal particle does not induce one or more immune side effects selected from the group consisting of inflammation, edema, allergy, and hypersensitivity when administered into the body.

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

19. A non-immunogenic polysaccharide cross-linked colloidal particle according to any one of claims 1 to 10, characterized in that the polysaccharide cross-linked colloidal particle does not act as a multivalent antigen having two or more epitopes unless it gels or aggregates at the site of administration through dosage adjustment.

20. A non-immunogenic polysaccharide cross-linked colloidal particle according to any one of claims 1 to 10, characterized in that the surface of the polysaccharide cross-linked colloidal particle is covalently or coordinately bonded to a nanoparticle, molecule, or metal ion through an amine functional group or a -COOH functional group derived from a cross-linking agent.

21. A non-immunogenic polysaccharide cross-linked colloidal particle characterized in that the number of drugs bound thereto (Drug-Particle Ratio, DPR) is controlled in accordance with the number of amine groups derived from the cross-linking agent in paragraph 20.

22. A non-immunogenic polysaccharide cross-linked colloidal particle characterized in that the polysaccharide cross-linked colloidal particle, modified with a ligand that targets a cell surface receptor through a cross-linking agent-derived amine functional group or a -COOH functional group, in claim 20, is maintained at the target site for at least 1 day, preferably at least 4 days.

23. A pharmaceutical composition characterized in that the non-immunogenic polysaccharide cross-linked colloidal particle of any one of claims 1 to 10 is used as a drug delivery vehicle or a lesion-targeting delivery platform for early diagnosis of inflammatory diseases.

24. A pharmaceutical composition characterized in that the non-immunogenic polysaccharide cross-linked colloidal particle of any one of claims 1 to 10 is used as a contrast agent for a quantitative indicator of an AI-based image analysis system.

25. A diagnostic composition characterized in that the non-immunogenic polysaccharide cross-linked colloidal particle of any one of claims 1 to 10 is used as a contrast agent capable of differentiating or staging inflammatory lesions.

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