Acid-resistant core-shell nanocomplex for oral use and mrcp t2-negative contrast using same

Abstract

The present invention relates to an orally administrable complex having an average hydrated particle diameter of 10 nm to 100 nm and comprising: a core composed of metal or metal oxide-based nanoparticle(s); and a shell disposed as an acid-resistant barrier layer on the surface of the core and coated with polysaccharide crosslinked colloidal particles which form multi-point coordination bonds with metal ions on the surface of the core and have an average hydrated particle diameter of 2 nm to 8 nm and a surface charge of -20 mV to 0 mV. The polysaccharide crosslinked 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 using a first crosslinking agent having an epoxide group, and (a) the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group are directly crosslinked and / or (b) two spatially adjacent functional groups modified by the first crosslinking agent are crosslinked intramolecularly and / or intermolecularly via a second crosslinking agent having two or more amine groups (-NH2) to form polysaccharide crosslinked particles, and (ii) the number of crosslinking agent-derived basic amine groups exposed on the surface is modified with a -COOH functional group so that the surface charge of the polysaccharide crosslinked colloidal particles is in the range of -20 mV to 0 mV. The orally administrable complex according to the present invention is a T2 MRI contrast agent that can be used as an active ingredient of a composition for diagnostic biliary and pancreatic imaging.

Description

Oral acid-resistant core-shell nanocomposite and MRCP T2-negative contrast using the same

[0001] The present invention relates to an oral T2 contrast agent that improves biliary-pancreatic duct (MRCP) contrast by treating the gastrointestinal lumen as a negative contrast in magnetic resonance imaging (MRI). Specifically, the invention relates to a core-shell type orally administered complex comprising a core made of metal or metal oxide-based nanoparticle(s) (e.g., SPION) and a shell made of polysaccharide cross-linked colloidal particles that form multivalent coordination bonds through metal ions on the core surface and multiple functional groups present on the surface. The polysaccharide cross-linked colloidal particles are designed such that their hydration average particle size is 2 nm to 8 nm and their surface charge is controlled to be -20 mV to 0 mV, and the final complex, which is coated with these particles on a core made of metal or metal oxide-based nanoparticle(s) (e.g., SPION), has a hydration average particle size in the range of 10 nm to 100 nm. This orally administered complex reduces or nulls the water signal of the stomach and / or duodenum, thereby inducing negative contrast in T2-weighted images; therefore, it can be used as an active ingredient in a composition for biliary and pancreatic duct imaging diagnostics for high-contrast imaging of the bile ducts and pancreatic ducts.

[0002] The present invention relates to a metal or metal oxide nanoparticle-based composition that can be orally administered for in vivo imaging diagnostics such as MRI or for location tracking, and to a core-shell oral formulation designed to provide a homogeneous and reproducible signal by simultaneously suppressing dissolution, aggregation, and precipitation in a highly acidic environment such as gastric juice.

[0003] Magnetic Resonance Imaging (MRI) is a technology that generates images by utilizing the magnetic properties of hydrogen protons within the human body. When radio frequency (RF) pulses are applied to protons aligned under a strong, uniform magnetic field, their alignment is disrupted; upon removal of the pulses, they return to their original state while releasing energy. Since the rate of realignment and the emitted energy vary depending on differences in the chemical environment surrounding the protons, signal differences are formed between tissues, allowing for a certain level of contrast to be obtained even without the use of contrast agents. Nevertheless, administering a contrast agent modulates the relaxation characteristics of protons within the microenvironment where the agent is distributed, further enhancing contrast between tissues.

[0004] In T2-weighted images, structures with high free water content appear relatively bright, so water signals in the lumen of the gastrointestinal tract (especially the stomach and duodenum) often interfere with the visibility of fluid columns such as the bile ducts and pancreatic ducts (Fig. 1a). Therefore, by using an orally administered negative contrast agent to reduce water signals in the lumen of the gastrointestinal tract, the anatomical structures of the bile ducts and pancreatic ducts can be visualized more clearly (Fig. 1b). Iron oxide-based nanoparticles (e.g., SPION) can provide a negative contrast effect by inducing local magnetic sensitivity differences through superparamagnetism, thereby promoting spin phase dephasing and shortening the T2 (or T2*) time.

[0005] Although SPIO agents such as Ferumoxide (Feridex) were commercialized in the past, their clinical application was limited due to rapid removal by the reticuloendothelial system and accumulation in specific organs. For example, superparamagnetic iron oxide (SPIO) agents with a diameter of approximately 50–200 nm are phagocytosed relatively quickly by the reticuloendothelial system and removed from the blood within a short time, and preferentially distributed to Kupffer cells in the liver, thereby reducing the signal intensity of normal liver parenchyma in T2 and T2*-weighted images.

[0006] The bile ducts and pancreatic ducts serve as pathways for digestive fluids, and cancer, inflammation, gallstones, pancreatic stones, and strictures are known to be major causes of biliary and pancreatic duct diseases. Imaging diagnostic methods for these diseases include endoscopic retrograde cholangiopancreatography (ERCP), ultrasound, and magnetic resonance cholangiopancreatography (MRCP). MRCP utilizes T2-weighted sequences to non-invasively visualize the bile and pancreatic duct systems, and bile columns with relatively long T2 relaxation times exhibit higher signal intensity than surrounding soft tissues. Sequences widely used in clinical practice include RARE (Rapid Acquisition with Relaxation Enhancement), FRFSE (Fast Recovery Fast Spin Echo), and HASTE (Half-Fourier Acquisition Single-Shot Turbo Spin Echo). MRCP is essential for the evaluation of various pancreatobiliary diseases, such as cholangiolithiasis, neoplasms of the biliary and pancreatic systems, congenital biliary malformations, chronic pancreatitis, primary sclerosing cholangitis, and postoperative biliary complications. With technological advancements, the accuracy and clinical role of MRCP are continuously expanding.

[0007] Meanwhile, functional MR cholangiography using gadoxetic acid (Gd-EOB-DTPA), a hepatocyte-specific T1 contrast agent, provides useful information for evaluating biliary leakage and strictures. Gadoxetic acid is absorbed by functioning hepatocytes, with approximately 50% excreted via the hepatobiliary pathway and the remainder excreted through the kidneys. After intravenous administration, T1-weighted images are acquired in dynamic phases (arterial, portal, and transitional phases) and the hepatobiliary phase (typically 20 minutes after administration, with 2–3 hour delayed imaging in some cases). Extrabiliary leakage of the active contrast agent is directly visualized as high signal, which aids in the accurate localization of the leakage site. However, delayed imaging is required for slow leakage; while it is advantageous for imaging the proximal bile duct adjacent to the liver, the signal weakens as it moves toward the distal common bile duct adjacent to the stomach and duodenum, which can make interpretation difficult; and there are limitations in imaging the pancreatic parenchyma. In addition, in patients with impaired liver function or hyperbilirubinemia, hepatocyte resorption and biliary excretion are reduced, which may limit the effectiveness of the technique.

[0008] Several technical barriers must be overcome to apply oral negative contrast agents to MRCP. The first is the preservation of magnetism in acidic environments. Iron oxide nanoparticles [regarding] Fe in gastric acid (pH 1–2). 2+ / Fe 3+As ions are released, crystallinity is impaired, and saturation magnetization and superparamagnetism are weakened, leading to a sharp decrease in T2 contrast effect. Second, there are issues regarding aggregation, precipitation, and image inhomogeneity. If surface charge and surface chemistry control are insufficient, inter-particle aggregation increases, and under the MRI magnetic field, magnetization-induced aggregation overlaps to form a precipitation layer, resulting in localized contrast effect and reduced effective concentration in the upper gastrointestinal tract. Third, non-specific interactions with mucus and enzymes reduce dispersion stability, causing physical properties to fluctuate from ingestion until image acquisition. Fourth, there is the difficulty in ensuring pharmacokinetic predictability. Since hydrated particle size and surface charge affect mucosal adhesion, pyloric passage, and small intestinal transit, it is necessary to reproducibly maintain stable T2 signal reduction or extinction for a certain period (e.g., more than 1 hour) during gastric retention. Fifth, management of free iron ions is required. Iron ions liberated by acidic leaching induce oxidation-reduction reactions that adversely affect dispersion stability, color, and attenuation coefficients (r2, etc.), so chelation or surface coordination fixation is required.

[0009] Therefore, oral T2 contrast agents must preserve the superparamagnetism of the core and suppress iron ion release even in the highly acidic environment of the stomach, and maintain homogeneous dispersion by preventing aggregation and precipitation under an external magnetic field. Furthermore, the hydration mean particle size and surface charge must be designed within an appropriate range to provide stable negative imaging during gastric retention, and chemical stability must be enhanced through coordination bonding / chelation using hydroxyl, amine, and carboxyl groups exposed on the surface. In addition, resistance to carbohydrate degrading enzymes and immunological inactivity must be secured, and formulation and dosage design can induce gallbladder contraction and bile secretion to relatively brighten bile duct signals. Ultimately, the development of core-shell oral formulations that simultaneously satisfy conflicting requirements—such as inhibition of acid release, prevention of magnetization-induced aggregation, enzyme and mucosal resistance, size and charge-based PK control, and free iron ion management—remains a key challenge for achieving homogeneous and predictable negative imaging in the gastric and duodenal lumens and improving contrast in biliary and pancreatic duct imaging.

[0010] Magnetic Resonance Cholangiopancreatography (MRCP) diagnoses lesions by utilizing the high signals of digestive fluids (main component: water) flowing through the bile and pancreatic ducts; however, since the lumens of the stomach and duodenum also consist of water, they appear very bright in T2-weighted images, blurring the boundaries of the bile and pancreatic ducts and degrading resolution. Currently, there are no oral T2-negative contrast agents available in clinical practice that can reliably suppress the water signals of the stomach and duodenum. Furthermore, metal oxide nanoparticles such as iron oxide (SPION) [expose] to Fe in gastric acid (pH 1–2). 2+ / Fe 3+ As it leaches out, crystallinity and superparamagnetism are weakened, and the T2 contrast effect decreases sharply. If surface charge and surface chemistry are inadequate, interparticle aggregation and magnetization-induced aggregation occur, leading to precipitation. Consequently, localized contrast appears only in areas adjacent to the precipitation layer, resulting in heterogeneous overall imaging. Furthermore, non-specific interactions with gastrointestinal mucus and enzymes reduce dispersion stability, causing a time-dependent decline in formulation performance.

[0011] The objective of the present invention is to provide an oral T2-negative contrast agent formulation that simultaneously resolves the above limitations and selectively reduces water signals in the lumen of the stomach and duodenum, thereby stably improving the visibility and contrast of the bile and pancreatic ducts. To this end, the -OH group of a polysaccharide is modified in an aqueous solvent with a first crosslinking agent having an epoxide group, and then an adjacent -OH or two adjacent modifying groups are crosslinked intramolecularly or intermolecularly with a second polyamine crosslinking agent to form a polysaccharide crosslinked particle. Subsequently, the surface basic amine number is partially substituted with -COOH to design a polysaccharide crosslinked colloid particle as a shell, with the surface charge precisely controlled to -20 mV to 0 mV. This shell forms multi-point coordination bonds with metal ions on the surface of a metal or metal oxide core to cover the entire surface, and maintains the hydration average particle size of the final complex in the range of 10 to 100 nm. As a result, the shell simultaneously implements functions such as a strongly bound anchor ligand that fixes the core in an acidic gastric environment, an acid-resistant barrier layer that blocks acid leaching, microenvironment control by maintaining zeta potential and hydration layer, inhibition of aggregation (colloid stabilization) that weakens van der Waals and magnetization interactions between particles, and inhibition of sedimentation (suspension stabilization) that lowers the sedimentation rate. The inventors confirmed that the core-shell structure can improve biliary-pancreatic duct contrast while maintaining a homogeneous contrast effect during a residence time in the stomach of 30 to 120 minutes.

[0012] A first aspect of the present invention comprises a core composed of metal or metal oxide-based nanoparticle(s); An oral administration complex having a hydration average particle size of 10 nm to 100 nm comprising a shell coated with polysaccharide crosslinked colloidal particles having a hydration average particle size of 2 nm to 8 nm and a surface charge of -20 mV to 0 mV, wherein the polysaccharide crosslinked colloidal particles are formed in an aqueous solvent by (i) modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, with a first crosslinking agent having an epoxide group, (a) directly between the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group, and / or (b) intramolecularly and / or intermolecularly crosslinking between two spatially adjacent functional groups modified by the first crosslinking agent through a second crosslinking agent having two or more amine groups (-NH2), thereby forming polysaccharide crosslinked particles, and (ii) exposed on the surface The present invention provides an orally administered complex characterized by a surface charge of polysaccharide crosslinked colloid particles within the range of -20 mV to 0 mV through modification of the number of basic amine groups derived from the crosslinking agent to -COOH functional groups.

[0013] For example, when administered orally, it induces gallbladder contraction and bile secretion, thereby exhibiting an auxiliary effect that relatively increases bile duct signals in MRCP. For example, even if the iron oxide-based nanoparticles of the oral administration complex become magnetized when exposed to an external magnetic field, the particles do not aggregate in the stomach due to the polysaccharide cross-linked colloidal particles coated on the core surface.

[0014] A second aspect of the present invention comprises a core composed of metal or metal oxide-based nanoparticle(s); A nanocomposite having a hydrated average particle size of 10 nm to 100 nm comprising a shell coated with polysaccharide crosslinked colloidal particles having a hydrated average particle size of 2 nm to 8 nm and a surface charge of -20 mV to 0 mV, which form multi-point coordination bonds with metal ions on the core surface, wherein the polysaccharide crosslinked colloidal particles are formed in an aqueous solvent by (i) modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, with a first crosslinking agent having an epoxide group, (a) directly between the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group, and / or (b) intramolecularly and / or intermolecularly crosslinking between two spatially adjacent functional groups modified by the first crosslinking agent through a second crosslinking agent having two or more amine groups (-NH2), thereby forming polysaccharide crosslinked particles, and (ii) the number of crosslinking agent-derived basic amine groups exposed on the surface is -COOH The present invention provides an acid-resistant nanocomposite designed such that, through modification with functional groups, the surface charge of the polysaccharide cross-linked colloidal particles is within the range of -20 mV to 0 mV, and the polysaccharide cross-linked colloidal particles simultaneously perform the functions of a strongly bonded anchor ligand, an acid-resistant barrier layer, an aggregation inhibitor, and a precipitation inhibitor.

[0015] For example, the oral administration complex of the first embodiment or the acid-resistant nanocomplex of the second embodiment may be manufactured such that it has a mononuclear core-shell structure or a multinuclear (bridged) core-shell structure that behaves as a single hydrated particle in a dynamic fluid environment, and some of the polysaccharide cross-linked colloidal particles coordinately bond to one or more cores to cross-link (share) between adjacent cores, so that the number of cores of the final oral administration complex is one or more, preferably four or more, and the hydrated average particle size is 10 nm to 100 nm.

[0016] For example, in the first embodiment of the oral administration complex or the second embodiment of the acid-resistant nanocomplex, some of the polysaccharide cross-linked colloidal particles that coordinate with the first core composed of metal or metal oxide-based nanoparticle(s) are simultaneously coordinated with the second core as well, so that the shell of the first core and the shell of the second core can share the same polysaccharide cross-linked particle(s).

[0017] A third aspect of the present invention provides an MRI contrast agent pharmaceutical composition comprising an acid-resistant nanocomposite of the second aspect.

[0018] A fourth aspect of the present invention provides a composition for diagnosing biliary and pancreatic duct imaging containing the oral administration complex of the first aspect as a contrast agent.

[0019] A fifth aspect of the present invention provides a method for imaging the bile duct or pancreatic duct in high contrast by orally administering the oral administration complex of the first aspect to a subject and performing T2-weighted MRI or MRCP while the oral administration complex remains in the stomach or duodenum to reduce gastrointestinal signals.

[0020] A sixth aspect of the present invention provides a method for preparing an acid-resistant nanocomposite of a second aspect, comprising the steps of: preparing a metal or metal oxide nanoparticle core suspension; forming polysaccharide cross-linked colloidal particles by modifying the -OH groups of a polysaccharide in an aqueous solvent with a first cross-linking agent having an epoxide group and cross-linking intramolecularly / intermolecularly with a second cross-linking agent of a divalent or higher polyamine, and then partially modifying the amine groups on the surface of the polysaccharide cross-linked colloidal particles with -COOH to adjust the zeta potential to -20 mV to 0 mV; and mixing the metal or metal oxide nanoparticle core suspension and the polysaccharide cross-linked colloidal particle solution having a zeta potential of -20 mV to 0 mV.

[0021] A seventh aspect of the present invention relates to a method for manufacturing an orally administered stabilizing modifier that induces the formation of an acid-resistant barrier, inhibition of inter-particle aggregation, and inhibition of precipitation under gastric pH 1 to 2 conditions, wherein (i) the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, is modified in an aqueous solvent by (i) modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, with a first crosslinking agent having an epoxide group, and (a) directly between the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group, and / or (b) intramolecularly and / or intermolecularly crosslinking between two spatially adjacent functional groups modified by the first crosslinking agent through a second crosslinking agent having two or more amine groups (-NH2), and (ii) the polysaccharide crosslinked colloidal particles, with the surface charge controlled to a range of -20 mV to 0 mV by modifying the number of crosslinking agent-derived basic amine groups exposed on the surface to -COOH functional groups, is used. Provides a use case.

[0022]

[0023] The present invention will be described below.

[0024] In this specification, 'oral administration' means ingesting an oral administration complex through the mouth and passing it through the gastrointestinal (GI) tract.

[0025] In this specification, the ‘polysaccharide crosslinked colloidal particle-based platform technology’ refers to a polysaccharide crosslinked colloidal particle having a surface charge in the range of -20 mV to 0 mV and a hydrated average particle size of 2 nm to 8 nm, wherein (i) a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, has an -OH functional group that is modified by a first crosslinking agent having an epoxide group, (a) a polysaccharide crosslinked particle formed by direct and / or (b) two spatially adjacent functional groups modified by the first crosslinking agent being crosslinked intramolecularly and / or intermolecularly through a second crosslinking agent having two or more amine groups (-NH2), and (ii) a polysaccharide crosslinked colloidal particle having a surface charge in the range of -20 mV to 0 mV and a hydrated average particle size of 2 nm to 8 nm, formed by modifying the number of basic amine groups of crosslinking agent originating from the surface to -COOH functional groups.

[0026] In this specification, 'strongly bound anchor ligand' refers to a metal ion on the core surface (e.g., Fe 2+ / Fe 3+It refers to a functional group that forms multidentate coordination bonds on the core-shell interface to prevent desorption and substitution from occurring even in acidic (pH 1-2), chloride ion, and protein competitive environments. For example, multiple donor groups such as -COO / -OH / amine on the surface of polysaccharide cross-linked colloid particles functionalized by epoxide-polyamine crosslinking and subsequent carboxylation simultaneously form chelate bonds (bi-, tri-, or higher denticity) with the core metal ion and the inner-sphere, thereby increasing the effective coordination number and overall stability constant (β). This multidentate coordination (i) suppresses the leaching of metal ions in gastric fluid and preserves core crystallinity and superparamagnetism, (ii) imparts resistance to ligand exchange and protonation to maintain r2 performance and interfacial adhesion even during prolonged contact, and (iii) enables the formation of an acid-resistant barrier layer that continuously covers the core surface. In addition, when a single ligand partially coordinates to two or more cores, it can bridge adjacent cores to stabilize a multicore (bridged) core-shell structure. As an example of indicative performance criteria, if the cumulative release of core metal ions is low (e.g., ≤5% relative to total metal) after 120 minutes of exposure to artificial gastric fluid (pH 1–2), and the r₂ attenuation efficiency and particle dispersion metrics are substantially maintained, the ligand is considered to be a strongly coupled anchor ligand of the present invention.

[0027] In this specification, the 'acid-resistant barrier layer' refers to a continuous, dense coating with a thickness of 2 to 8 nm (based on hydration) formed by polysaccharide cross-linked colloidal particles disposed on the surface of a metal or metal oxide nanoparticle core forming multi-point coordination bonds with metal ions on the core surface. This layer (i) forms a strongly bonded anchor ligand network with the core (preferably, the lattice) by the coordination fixation of surface functional groups such as -COO / -OH / amine, (ii) inhibits the microdiffusion of protons and chloride ions into a hydrated polymeric medium formed by the cross-linking network, which is a local environment formed by the bound water on the particle surface and the polymer network, thereby suppressing the dissolution of the core and crystallization damage under acidic conditions (pH 1 to 2), and (iii) controls the surface charge to a range of -20 mV to 0 mV and provides steric hindrance to suppress inter-particle contact, aggregation, and magnetization-induced precipitation. As a result, the superparamagnetic and r2 attenuation characteristics of the core are maintained even in highly acidic environments such as gastric juice, and spatial and temporal homogeneity in suspension / solution states is ensured. The barrier layer includes both a single-layer coating form and a multilayer stack ([polysaccharide cross-linked colloidal particle-metal ion] repeating), and is applied to both single-core structures and multi-core (bridged) core-shell structures.

[0028] In this specification, the term 'anti-agglomeration and anti-sedimentation function' refers to integrated stabilization mechanisms designed so that core-shell nanocomposites do not undergo particle-particle bonding / cluster formation (agglomeration) and gravitational separation (sedimentation) over time in highly ionic, acidic environments such as gastric juice (pH 1–2) and / or under an MRI magnetic field. For example, (i) the -COO of the shell - It includes (ii) electrical repulsion resulting from the control of the / -OH / amine functional group arrangement and surface charge (ζ -20 mV ~ 0 mV), (iii) electrosteric stabilization by a steric and hydration barrier provided by a hydrated polysaccharide crosslinking network with a thickness of 2–8 nm, (iii) suspension stabilization that reduces Stokes settling velocity through viscosity modulation and hydration layer formation, and (iv) magnetic-field stability that suppresses magnetization-induced dipolar agglomeration due to the absence of remanent magnetization of the superparamagnetic core and steric hindrance of the shell. Additionally, it includes -COO on the shell surface - / amine / OH chelates trace-free metal ions to prevent cross-linking (bridging) agglomeration and minimizes nonspecific adsorption with gastric mucus / proteins. As an example of performance indicators, the “agglomeration inhibition and precipitation inhibition function” of the present invention is satisfied if it is confirmed that there is no visible precipitation upon 120 minutes of exposure to artificial gastric fluid (pH 1–2), no significant change in DLS effective diameter and PDI, r2 retention rate ≥80%, stability of turbidity / Turbiscan indicators, and no significant change in dynamic light scattering distribution before and after magnetic field (exposure to 1.5–3.0 T). This function ensures a homogeneous dispersion state and temporal signal reproducibility throughout the stomach and duodenum, thereby stably securing spatial homogeneity and image contrast in T2-negative contrast imaging.

[0029] In this specification, a ‘multicore (bridged) core-shell structure’ refers to a structure designed to behave as a single hydrated particle in a dynamic fluid environment, wherein a core composed of two or more (preferably four or more) metal or metal oxide-based nanoparticles is connected (bridged) to one another by polysaccharide cross-linked colloidal particles that function as a single shell, as exemplified in FIG. 2c. Each polysaccharide cross-linked colloidal particle bridges adjacent cores by forming multi-point coordination bonds with two or more core surface metal ions through surface functional groups (e.g., -COOH), and a network of these covalent bonds forms a continuous shell that encloses the entire core group. Consequently, the resulting average hydrated particle size is in the range of 10 to 100 nm, which is distinct from the crystal size of the individual core (e.g., Fe3O4, approximately 8 nm). Unlike a single-core core-shell structure, this structure simultaneously provides functional advantages such as (i) the formation of an acid-resistant barrier layer by a multidentate coordination network, (ii) colloid stabilization by surface charge (-20 to 0 mV) and steric hindrance, and (iii) suppression of magnetization-induced aggregation with improved r2 relaxation. For example, the preparation involves preparing a metal or metal oxide nanoparticle core suspension. Separately, under aqueous conditions, the -OH groups of the polysaccharide are intramolecularly / intermolecularly crosslinked with an epoxide (first crosslinking agent) and a divalent or higher polyamine (second crosslinking agent) to form polysaccharide crosslinked colloid particles, and some of the surface amines are substituted with -COOH to control the zeta potential to -20 to 0 mV. When the two suspensions are mixed and stirred, the -COO on the particle surface -A multinuclear (bridging) core-shell structure is self-assembled as isofunctional groups form multi-point coordination bonds with metal ions on the core surface, thereby cross-linking (shared) adjacent cores. Hydration diameter (10–100 nm) and bridge density can be controlled by the core-to-particle molar ratio, pH / ionic strength, and mixing time, and unbound components can be removed by dialysis / filtration to obtain an acid-resistant nanocomposite that is stable to gastric acid (pH 1–2). During measurement, the average size as a single hydrated particle is observed in dynamic light scattering (DLS), while a morphology in which multiple cores are encapsulated into a common shell is identified in TEM; the bridging multinuclear characteristics can be confirmed through the combination of these two data. This structure is particularly advantageous for achieving uniform T2-negative contrast in MRCP by preserving the magnetism of the core and suppressing aggregation and precipitation under exposure to gastric acid (pH 1–2) in oral formulations.

[0030] In this specification, the term 'acid-resistant nanocomplex' refers to a core-shell structure using the aforementioned 'polysaccharide cross-linked colloidal particle-based platform technology,' wherein the shell is designed to function as an acid-resistant barrier layer. This complex may be configured to (i) preserve core magnetism and suppress metal ion release through multi-point coordination bonding at the core-shell interface, (ii) suppress aggregation / precipitation and maintain suspension stability during gastric retention through the surface charge, cross-linking network, and hydration layer of the shell, and (iii) improve MRCP contrast by uniformly reducing gastric and duodenal moisture signals through T2 (or T2*) shortening. Acid-resistant nanocomposites include both mononuclear core-shell and multinuclear (bridging) core-shell variations, and in both cases, the size, surface charge, degree of crosslinking, and coordination characteristics with metal ions can be adjusted to maintain magnetic, suspension, and colloidal stability for a certain period of time (e.g., 30 to 120 minutes) in a gastric cavity pH of 1 to 2. Accordingly, the acid-resistant nanocomposites of the present invention provide a composite-based platform technology for oral administration.

[0031] In this specification, 'oral nanocomplex-based platform technology' refers to a technical framework that enables the fabrication and application of acid-resistant core-shell nanocomposites using shared design rules and modular processes. The core components are (a) a metal or metal oxide-based nanoparticle core (including single-domain superparamagnetic crystals or amorphous particles) and (b) a polysaccharide crosslinked colloidal particle shell with a hydration thickness of 2 to 8 nm, prepared by epoxide-polyamine crosslinking and partial carboxylation in aqueous media, with a surface charge controlled to -20 mV to 0 mV. The shell forms multi-point coordination bonds with the core surface metal ions to act as a strongly bonded anchor ligand and an acid-resistant barrier layer, and is designed to simultaneously achieve inhibition of aggregation and precipitation through charge, steric hindrance, and the hydration layer. This platform is applicable to both mononuclear core-shell and multinuclear (bridging) core-shell variants, in which case a subset of polysaccharide crosslinked colloidal particles are coordinated like bridges to two or more cores, maintaining a hydration average particle size in the range of 10 to 100 nm. Fabrication is standardized into a modular process of “core suspension preparation → synthesis of polysaccharide crosslinked colloidal particles and ζ-potential control → spontaneous coordination coating by mixing the two suspensions (multilayer stacking if necessary),” which ensures reproducibility of r2 performance and particle size distribution between batches. Performance objectives are defined as r2 retention rate (e.g., ≥80%) upon 120 minutes of exposure to artificial gastric fluid (pH 1–2), absence of visible precipitation, DLS size and PDI stability, and inhibition of magnetization-induced aggregation under a magnetic field of 1.5–3.0 T.The formulation can be extended to oral suspensions or reconstituted powders / concentrates, and the pH, osmolality, viscosity, and shelf life ranges are adjusted to pharmacopoeial standards. The platform is primarily intended for negative contrast applications that enhance MRCP contrast by reducing the water signal through uniform T2 (or T2*) shortening during gastric and duodenal transit, but it offers scalability to be customized for various clinical scenarios using parameters such as core composition, shell thickness, surface charge, degree of crosslinking, and viscosity control.

[0032] The inventors developed an MRCP-specialized T2 MRI contrast agent that effectively suppresses water signals in the stomach and duodenum by applying the orally administered complex-based platform technology of the present invention for high-resolution imaging and accurate diagnosis of the bile ducts and pancreatic ducts (Examples 1–13). Specifically, a core-shell complex (total hydration average particle size 10–100 nm) was designed comprising a core made of iron oxide-based nanoparticle(s) and an acid-resistant barrier shell composed of polysaccharide cross-linked colloidal particles (hydration average particle size 2–8 nm, surface charge -20 mV–0 mV) that form multi-point coordination bonds with iron ions on the core surface. Here, the polysaccharide cross-linked colloidal particles forming the shell are dextran-based nanostructures synthesized using a polysaccharide cross-linked colloidal particle-based platform process (Example 1), and an MRI T2 contrast agent (INV-003) was prepared using this (Fig. 2 and Examples 2–8). The contrast agent did not degrade while maintaining a stable T2-negative contrast effect for more than 1 hour in the gastric and / or duodenal environment, and no significant toxicity was observed in mouse toxicity tests with both intravenous and oral administration (Examples 10, 12).

[0033] INV-003 is an oral T2 contrast agent in which iron oxide-based particles (cores) are surface-modified into polysaccharide cross-linked colloidal particles (hydrated average particle size 2–8 nm, surface charge -20 mV–0 mV). It enhances MRCP contrast by effectively nulling water signals within the stomach and duodenum during T2-weighted MRI. Although iron oxide is prone to ionization in acidic conditions and loss of T2 effect, it was confirmed that INV-003 stably maintains its T2 contrast effect for more than one hour under gastric acid (pH 1.2) conditions (Example 11-1, Fig. 13). Furthermore, at oral dosages of 5–200 mL, it demonstrated physiological benefits by inducing gallbladder contraction and bile secretion, thereby further amplifying high signals in the bile ducts (Example 11-2, Fig. 14).

[0034] The present invention provides a shell in which, under aqueous conditions, (i) the -OH groups of a polysaccharide are modified with an epoxide first crosslinking agent, (ii) intramolecular and intermolecular crosslinking is performed with a polyvalent amine second crosslinking agent to form crosslinked particles of several nm, and (iii) a portion of the surface amine is converted to -COOH to precisely control the zeta potential to -20 mV to 0 mV. This shell forms multi-point coordination bonds with metal ions on the core surface to cover the entire surface of the core, and maintains the hydration average particle size of the final complex in the range of 10-100 nm (Figs. 2b, 2c). As a result, the shell simultaneously performs (a) strong-bonded anchor ligands, (b) an acid-resistant barrier layer, (c) microenvironment control, (d) inhibition of aggregation (colloid stabilization), and (e) inhibition of precipitation (suspension stabilization) in gastric fluid.

[0035] Polysaccharide cross-linked colloidal particles form multi-point coordination bonds with core metal ions through surface -COOH / -OH / residual -NH2 groups, while simultaneously chelating free metal ions in situ to suppress dissolution and magnetic degradation. This structural and surface-chemical design preserves dispersibility and r2 performance even at pH 1.2 and blocks magnetization-induced aggregation and bridge aggregation under high ionic intensity environments and magnetic fields (Fig. 10). In clinical application, signals throughout the gastric lumen are uniformly degraded for 30–60 minutes or more, clarifying the bright T2 signals of the bile and pancreatic ducts (Fig. 14b), and provides a superior unit-concentration contrast effect compared to conventional formulations at the same concentration. Furthermore, since this complex acts primarily locally in the gastrointestinal tract and systemic absorption is minimal, no changes in liver or blood T2* indicators were observed before or after administration (Fig. 20). In summary, with (i) a cross-linked shell of several nm, (ii) a surface charge of -20 to 0 mV, (iii) multi-point coordination with metal ions, and (iv) a combined design of 10 to 100 nm particle sizes, INV-003 uniformly knurls gastric and duodenal moisture signals for a long time, significantly improving the signal-to-noise ratio / contrast-to-noise ratio of MRCP.

[0036] One embodiment of the present invention (Fig. 2c) is a multinuclear (bridged) core-shell structure designed such that multiple superparamagnetic iron oxide cores of 7-9 nm are individually coated by polysaccharide cross-linked colloidal particles with a hydrated particle size of 2-8 nm, and the coating layers function as a continuous single shell through the interpenetration and physical interlocking of the hydration and cross-linking brushes. During manufacturing, the -COO on the surface of the polysaccharide cross-linked colloidal particles -The / -OH / and residual -NH2 functional groups form multi-point coordinate bonds only with surface Fe ions of each core, forming an acid-resistant mosaic layer that does not detach even under acid-base conditions (pH 1-2). Here, a single colloidal particle is coordinated to two or more cores simultaneously, but the process of chemically cross-linking colloidal particles to form additional layers is not included. The multi-nucleated arrangement is obtained because the shells of individual cores overlap each other and behave like a continuous layer; in TEM, the cores are identified as approximately 8 nm, and in DLS, an effective size of 40-50 nm (e.g., 45±5 nm) reflecting the hydration shell and continuous shell effects is observed (Fig. 6). This structure (i) suppresses metal ion leaching through an acid-resistant barrier, (ii) simultaneously suppresses bridge / magnetization-induced aggregation through -20 to 0 mV surface charges and steric repulsion, and (iii) maintains the structure even in high-ionic steel (up to 1 M NaCl). As a result, it remains in a uniform suspension within the stomach and duodenum, efficiently shortening T2 (or T2*) relaxation to numb fluid signals and selectively increasing bile duct and pancreatic duct contrast.

[0037] Meanwhile, a complex according to another embodiment of the present invention comprises a core composed of metal or metal oxide-based nanoparticle(s) and a shell disposed on the surface of the core as an acid-resistant coating layer. The shell comprises polysaccharide cross-linked colloidal particles having a hydration average particle size of 2 to 8 nm and a surface charge of -20 mV to 0 mV, and metal ions (e.g., Fe 3+ Repeating units [polysaccharide cross-linked colloidal particles-metal ions] alternately stacked on the core surface mIt can be composed of (m = 1~20). It includes a process of creating additional layers through chemical crosslinking between colloidal particles. By controlling the degree of repeated stacking (m), core:colloid ratio, pH, and ionic strength, the hydrated average particle size of the final composite can be controlled to a range of 10~100 nm, and it can be designed to suppress metal ion leaching, suppress aggregation / precipitation, and maintain homogeneous dispersion in an acidic environment (pH 1~2).

[0038]

[0039] [Core composed of metal or metal oxide-based nanoparticle(s)]

[0040] In this specification, 'core' refers to nanoparticle(s) composed of a metal or metal oxide, and includes crystalline single-domain nanocrystals (preferably superparamagnetic) as well as amorphous particle(s). The core functions as a central structure in which an acid-resistant barrier layer is formed through multi-point coordination bonding between polysaccharide cross-linked colloidal particles and metal ions on the core surface. The core may be a metal (Ga, Au, Ag, Pt, Cu, Ni, Co, Mn, Gd, etc.) or metal oxide (e.g., Fe3O4, γ-Fe2O3, MnO / Mn3O4, CoFe2O4, MnFe2O4, Gd2O3, etc.) nanoparticle. Preferably, a spinel-type or mixed oxide structure ferrite (MFe2O4, M=Mn, Co, Ni, Zn, etc.) or magnetite / maghemite-based iron oxide may be used. The core size is preferably 4 to 15 nm (e.g., 6 to 10 nm), and in the case of crystallinity, it is desirable to exhibit single-domain superparamagnetism so that no residual magnetization remains substantially when the external magnetic field is removed. The amorphous core has metal ion sites capable of coordination on its surface (e.g., Fe) even if the crystal lattice does not possess long-range order. 2+ / Fe 3+It can form stable multi-point coordination with polysaccharide cross-linked colloidal particles through interaction with amorphous surface defects / functional groups, and can be designed to provide sufficient changes in magnetism / magnetic sensitivity for T2 (or T2*) attenuation depending on composition, size, and oxidation state. The synthesis method is not limited to co-precipitation, microemulsion, pyrolysis, reduction / oxidation processes, etc., and it is desirable to satisfy a narrow size distribution (relative standard deviation ≤ 20%) and high chemical purity (low content of free metal ions and salt impurities) regardless of whether it is crystalline or amorphous. The present invention includes both mononuclear core (one nanoparticle) and multinuclear (bridging) core-shell configurations, and in the latter case, it is designed so that the hydration average particle size of the final core-shell composite is maintained in the range of 10 to 100 nm even if the polysaccharide cross-linked colloidal particles cross-link and share two or more cores. This core definition encompasses surface coordination capabilities and physical property requirements to ensure magnetic retention (r2 performance), inhibition of metal ion leaching, and colloid / suspension stability even in strong acidic conditions (pH 1~2) within the stomach.

[0041] The core of the present invention may be a metal or metal oxide nanoparticle designed to simultaneously satisfy T2-negative contrast imaging efficiency and gastric durability. Among metal oxides, magnetite (Fe3O4) or maghemite (γ-Fe2O3) is most preferred, and spinel-type ferrites such as MnFe2O4, CoFe2O4, and NiFe2O4 may also be used as needed. These materials combine high magnetization, low coercivity, and chemical stability, causing a strong difference in local magnetic sensitivity in an external magnetic field, while leaving no residual magnetization when the magnetic field is removed, thereby minimizing permanent aggregation between particles. To achieve superparamagnetism, the core diameter is typically limited to a range of 5 to 15 nm, preferably 6 to 10 nm; this is a condition to ensure single-domain behavior at room temperature, coercivity ≈ 0, and residual magnetization ≈ 0. As the core size increases, r2 increases, but the risk of aggregation and sedimentation due to residual magnetization also increases; therefore, in this invention, the size is selected at the balance point of r2 / particle stability, considering the combination with the shell.

[0042] Manufacturing can be carried out by one or a combination of coprecipitation, microemulsion, pyrolysis, and hydrothermal synthesis, considering process ease and consistency. In the coprecipitation method, Fe 3+ / Fe 2+After mixing (e.g., FeCl3·6H2O and FeCl2·4H2O) in a molar ratio of 2:1, a base (NaOH, NH4OH, TMAOH, etc.) is added under a nitrogen or argon atmosphere to control the nucleation and growth stages at 60–90°C. Target size and distribution can be obtained by finely controlling pH, ionic strength, and temperature, and long-term oxidative stability can be enhanced by partially oxidizing the magnetite immediately after synthesis to maghemite through mild oxygen treatment. The microemulsion method achieves a narrow distribution by inducing homogeneous nucleation within a limited reaction compartment inside a reverse micelle, while the pyrolysis method provides highly crystalline monodisperse nanocrystals by decomposing iron oleate / acetylacetonate precursors at high temperatures in a high-boiling point solvent (octadecene, etc.). Hydrothermal / solvothermal processes provide highly crystalline particles in an aqueous system and are advantageous for process scale-up. Any process follows the nucleation-growth-stopping phase (LaMer mechanism), and the average diameter, crystal phase, and surface -OH density are precisely controlled by adjusting the reaction time and surfactant concentration (oleic acid, oleylamine, citric acid, etc.).

[0043] After synthesis, the core is purified by magnetic separation-washing (ethanol / water), dialysis, or ultrafiltration to remove free iron ions and low molecular weight impurities. Particles prepared in the organic phase are converted into an aqueous dispersion through surface ligand exchange and capped with citric acid, phosphate, and polycarboxylate to secure metal coordination sites on the surface. The final aqueous core is treated to maintain a stable dispersion state at neutral (pH 7 ± 0.5) and to leave coordination vacancies capable of binding to metal centers on the surface, thereby preparing it to form multi-point coordination bonds with the polysaccharide cross-linked colloid particles of the present invention. This surface coordination capability is critical for the shell to be continuously formed across the front of the core to form an acid-resistant barrier layer.

[0044] Magnetic properties are evaluated using a Vibrating Sample Magnetometer (VSM) or SQUID, and saturation magnetization (M s) is determined by material, size, and crystallinity. Based on magnetite, M at 6–10 nm s Although lower relative to the bulk, it is sufficient for T2 attenuation, and coercivity (Hc) and remanent magnetization (Mr) must be maintained near the measurement limit. For MRI performance, a combination with a high r2 at 3 T and a sufficiently large r2 / r1 ratio is advantageous; while increasing core size tends to increase r2, the optimal point is selected considering the trade-off with the risk of intragastric deposition. Core size and distribution are determined by TEM / DLS, crystal phase by XRD / SAED, and Fe by XPS / Mossbauer. 2+ / Fe 3+ Reproducibility is guaranteed by checking the rain and oxidation state.

[0045] In terms of safety, residual salts and free iron can increase gastric reactivity and mucosal irritation, so the residual amount of metal ions is strictly controlled. In addition, biological impurities such as endotoxins are removed through purification, filtration, and sterilization processes. The core thus manufactured, purified, and characterized is firmly bonded to a shell coated with the polysaccharide cross-linked colloid particles of the present invention via multi-point coordination, forming a core-shell structure that preserves crystallinity, inhibits iron ion leaching, and suppresses aggregation under a magnetic field even in an acidic gastric environment. Consequently, the core simultaneously achieves high T2 attenuation efficiency and excellent suspension stability, functioning as a foundational element that effectively suppresses gastric and duodenal signals in MRCP and maximizes the selective visibility of the biliary and pancreatic ducts.

[0046] The polysaccharide cross-linked colloidal particles of the present invention serve as a shell designed to simultaneously ensure the chemical and physical stability of an iron oxide-based nanoparticle core in an oral administration environment. They are a key component that maintains the superparamagnetism and dispersibility of the core even under conditions of strong gastric acidity (pH 1–2) and magnetic field exposure. The polysaccharide cross-linked colloidal particles, controlled with a hydration average particle size of 2–8 nm and a surface charge of -20 mV–0 mV, form multi-point coordination bonds with metal ions on the core surface to act as “strongly bonded anchor ligands.” By constructing an acid-resistant barrier layer with a cross-linking network of 2–8 nm thickness, they delay the penetration of proton and chloride ions, thereby preventing the leaching of the core (Fe 2+ / Fe 3+ Inhibits (release) and crystal damage. -COO exposed on the surface -The / -OH / amine functional group maintains the zeta potential in a weak or near-neutral range, providing electrostatic repulsion and steric hindrance, thereby simultaneously suppressing magnetization-induced aggregation and precipitation caused by magnetic dipole interactions. This maintains the stability of the suspension colloid during gastric lumen retention of 30 to 120 minutes, enabling spatially homogeneous T2-negative contrast imaging. Furthermore, the polysaccharide cross-linked colloid particles possess resistance to non-specific interactions with carbohydrate-degrading enzymes and gastric mucus components, thereby minimizing surface property deformation due to protein adsorption and time-dependent heterogeneity. Multiple ligand groups (Fig. 2a) distributed in the cross-linking network to form multidentate coordinate bonds additionally chelate trace amounts of free metal ions, preventing color changes or r2 degradation and enhancing the quality stability of the formulation during storage and use. Precise control of the size and surface charge of polysaccharide cross-linked colloid particles maintains the hydration average particle size of the entire complex in the range of 10 to 100 nm, thereby inhibiting mucosal penetration and systemic absorption, and providing a safe profile centered on local action in the gastric and duodenal regions. In short, the polysaccharide cross-linked colloid particle shell of the present invention integrates four functions: coordination fixation (acid-resistant barrier), colloid / suspension stabilization (inhibition of aggregation and sedimentation), enzyme / mucus resistance, and free iron ion management. By preserving the superparamagnetism of the SPION core upon oral administration and achieving highly reproducible T2 signal reduction throughout the gastric cavity, it directly contributes to enhancement against MRCP.

[0047]

[0048] [Oral Contrast Agent]

[0049] The gastrointestinal tract contains various enzymes and an acidic environment designed to break down ingested substances. Dextran, a complex polysaccharide, is easily broken down by this digestive process. Enzymes such as amylase and other carbohydrases can break dextran into smaller pieces. The acidic environment of the stomach can also contribute to the breakdown of dextran molecules.

[0050] Amylase is an enzyme that breaks down complex carbohydrates. While amylase primarily acts on starch, it can also affect dextran to some extent. Amylase and other carbohydrate-degrading enzymes generally work by hydrolyzing the glycosidic bonds between sugar units in dextran molecules. The action of these enzymes can break down dextran into smaller oligosaccharides or individual glucose units.

[0051] The pH of the stomach is generally very low, ranging from 1.2 to 3.5. This acidic environment can trigger a chemical reaction called acid hydrolysis. The high concentration of hydrogen ions (H+) in stomach acid can catalyze the breakdown of glycosidic bonds in dextran. This acidic environment can weaken and potentially break the bonds between the sugar units of a dextran molecule. The extent of this degradation can vary depending on factors such as the specific structure and molecular weight of the dextran, as well as the time it spends in the stomach.

[0052] Enzymatic and acidic degradation processes can act simultaneously. Since acid partially breaks down the dextran structure, it may become more sensitive to enzymatic action. Conversely, if enzymes break down dextran into smaller fragments, it may become more vulnerable to acid hydrolysis. The presence of food or other substances in the gastrointestinal tract affects both pH and enzyme activity, potentially altering the degradation process.

[0053] The intestinal epithelium selectively chooses which molecules are delivered into the bloodstream. Large, intact dextran molecules may have difficulty passing through the intestinal barrier. Only smaller fragments or small portions of the original dextran molecules may be absorbed. Factors such as transit time, pH levels, and enzyme concentrations can affect the fate of orally administered dextran.

[0054] Accordingly, so that the polysaccharide cross-linked colloid particles coated on the core surface of the oral administration complex of the present invention are not hydrolyzed by carbohydrases and simultaneously perform the functions of 'strongly bound anchor ligand', 'acid-resistant barrier layer', 'aggregation inhibition', and 'precipitation inhibition' in the stomach, in an aqueous solvent, (i) the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, is modified with a first crosslinking agent having an epoxide group; (a) intramolecular and / or intermolecular crosslinking is formed between the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group, and (b) two spatially adjacent functional groups modified by the first crosslinking agent are crosslinked through a second crosslinking agent having two or more amine groups (-NH2) to form polysaccharide cross-linked colloids; and (ii) the number of basic amine groups derived from the crosslinking agent exposed on the surface is modified to -COOH functional groups to form polysaccharide cross-linked colloids. It is characterized by the fact that the surface charge of the particle is within the range of -20 mV to 0 mV.

[0055] Meanwhile, the method for preparing an oral administration complex of the present invention comprises the steps of: preparing a metal or metal oxide nanoparticle core suspension; forming polysaccharide cross-linked colloidal particles by modifying the -OH group of a polysaccharide in an aqueous solvent with a first cross-linking agent having an epoxide group and cross-linking intramolecularly / intermolecularly with a second cross-linking agent of a divalent or higher polyamine, and then partially modifying the amine group on the surface of the polysaccharide cross-linked colloidal particles with -COOH to adjust the zeta potential to -20 mV to 0 mV to prepare a polysaccharide cross-linked colloidal particle colloidal solution; and mixing the metal or metal oxide nanoparticle core suspension and the polysaccharide cross-linked colloidal particle colloidal solution having a zeta potential of -20 mV to 0 mV.

[0056] Polysaccharide crosslinked colloid particles are synthesized in an aqueous solvent. Specifically, the -OH functional groups of linear, branched, or cyclic polysaccharides, or monosaccharides which are their building blocks, are partially modified with a first crosslinking agent having an epoxide group, and then intramolecular and / or intermolecular crosslinking is performed by (a) direct crosslinking between the functional groups modified by the first crosslinking agent and spatially adjacent non-modified -OH functional groups, or (b) connecting two spatially adjacent modified groups with a second crosslinking agent having two or more primary / secondary amine groups, thereby forming a uniform crosslinked colloid with a size of 2 to 8 nm. Subsequently, some or all of the basic amine groups derived from the crosslinking agent remaining on the surface are reductively modified to -COOH functional groups by organic acid anhydride treatment, etc., and finally, the zeta potential of the polysaccharide crosslinked colloid is precisely controlled to a range of -20 mV to 0 mV. These negatively charged weakly acidic surfaces inhibit inter-particle bridge aggregation even at high salt concentrations in gastric juice, through the combination of electrostatic repulsion caused by the electric double layer and the steric hindrance effect of cross-linked polysaccharides.

[0057] When the polysaccharide cross-linked colloidal particles prepared as described above come into contact with the surface of a metal or metal oxide core, the polyvalent -OH of the polysaccharide chains and the introduced -COO - Functional groups such as (carboxylates) and residual -NH2 (amines) form multi-point coordination bonds with surface metal ions, forming a 'strongly bonded anchor ligand' layer several nanometers thick. Unlike single coordination or adsorption coatings, this multi-point coordination exhibits a significantly slower dissociation rate, and [Cl - It stabilizes the core-shell bond even under ~0.1 M conditions. At the same time, the -OH / -COO remaining on the shell surface -The / -NH2 functional group inhibits the diffusion into the solution phase by chelating and coordinating free metal ions that may be released in trace amounts under gastric acid conditions. As a result, lattice damage to the metal oxide core (e.g., iron oxide) and the consequent reduction in saturation magnetization, loss of superparamagnetism, and degradation of the T2 contrast effect are suppressed. Furthermore, even if the core is partially magnetized under a gradient magnetic field or a strong magnetic field of an MR scanner, the surface charge and steric hindrance of the shell block magnetization-induced aggregation caused by dipole interactions, and suspension stability is ensured by lowering the sedimentation velocity through average density and size design (10–100 nm). The combination of these 'acid-resistant barrier layer' and 'microenvironment control' functions ensures the uniform distribution of the complex throughout the gastric cavity and maintains a continuous nulling effect without phase separation over time.

[0058] The above mechanism of action is demonstrated by the results of the large animal (MRCP) study in Example 13. When INV-003 was orally administered to miniature pigs, the SNR of the gastric cavity ROI and the CNR relative to the liver decreased by tens of times compared to before administration (e.g., at 0.40 mg Fe / mL and 0.125–0.25 mg Fe / kg, the SNR decreased from hundreds to single digits, and the CNR decreased to near zero or negative), resulting in spatially homogeneous nulling of gastric and duodenal water signals. In qualitative evaluation, the visibility of the entire bile duct pathway improved to Good–Excellent, and reading interference caused by high gastrointestinal signals decreased to a level of ‘none / mild’. Under the same conditions, the liver T2* maps were 4.6±0.4 ms immediately after administration, 4.4±0.4 ms, and 4.3±0.5 ms on day 7 and day 13, respectively, showing no significant change (repeated measures statistic p>0.48), which supports the fact that the complex does not significantly accumulate in the liver. Furthermore, in the repeated oral toxicity screening of Beagle in Example 12, no unexpected death, changes in clinical symptoms, changes in body weight and feeding, hematological and biochemical abnormalities, necropsy, or changes in organ weight were observed up to high doses of up to 40 mg Fe / kg, confirming that the shell of this design extends the safety margin centered on local intestinal action.

[0059] The oral administration complex of the present invention comprises a core composed of metal or metal oxide-based nanoparticle(s) and a shell composed of polysaccharide cross-linked colloidal particles that form multi-point coordination bonds with surface metal ions of the core. The polysaccharide cross-linked colloidal particles constituting the shell are designed to have a hydration average particle size of 2 nm to 8 nm and a surface charge of -20 mV to 0 mV, thereby maintaining a colloidal state without aggregation or precipitation even in a highly acidic, high-ionic-strength gastric fluid environment. The hydration average particle size of the entire complex, in which the core and shell are combined, is 10 nm to 100 nm, which is advantageous for retention and uniform dispersion within the gastric cavity upon oral administration and stably induces T2 (or T2*) signal inhibition in the aqueous environment of the stomach and duodenum. For example, the oral administration complex of the present invention can maintain suspension stability without aggregation or precipitation for a retention time of 30 to 120 minutes in the stomach. Specifically, it may exhibit suspension stability in which the change in the average hydration particle size is maintained at 20% or less even under a dynamic magnetic field during a residence time of 30 to 120 minutes in the stomach.

[0060] Therefore, the orally administered complex of the present invention acts as a contrast agent and can provide more detailed information about disease processes at the tissue composition, function, or molecular level, as well as anatomical images of the accumulated site. MRI contrast agents are classified into paramagnetic and superparamagnetic agents depending on their effect on the magnetic field. Paramagnetic agents exhibit a dominant T1 attenuation effect, appearing as bright signals in T1-weighted images, while superparamagnetic agents exhibit a dominant T2 attenuation effect, causing the signal to darken in T2 images. The orally administered complex of the present invention is designed to act as a contrast agent to influence the relaxation times (T1 and T2) of hydrogen protons in surrounding water molecules, thereby enhancing the contrast in MR images between the target tissue and surrounding tissues (enhanced magnetic properties). The performance criteria for the orally administered complex of the present invention are, for example, as follows: free metal ion release in artificial gastric fluid (USP, pH 1.2, 37 ℃) for 120 minutes ≤ 5% of total metal, average change in hydration diameter ≤ 20%, no visible precipitation, r2 retention rate (1.5~3.0 T) ≥ 80%, and secondary (aggregation) peak area in dynamic light scattering under external magnetic field exposure ≤ 10% of the initial.

[0061] The structure of the oral contrast agent of the present invention operates regardless of the type of core metal. For example, the core may be a magnetic metal oxide such as iron oxide (Fe3O4 / γ-Fe2O3), a non-magnetic or weakly magnetic metal oxide such as manganese oxide, copper oxide, or cobalt oxide, or even metal nanoparticles such as gold, silver, copper, cobalt, or nickel, provided that the same barrier and stabilization effects are imparted by the coordination bonding of polysaccharide cross-linked colloid functional groups to metal ions or metal-oxygen coordination sites on the surface. When the core is magnetic, T2 (or T2*) signal attenuation is maximized to selectively suppress gastric lumen water signals, and even when the core is non-magnetic, formulation homogeneity and safety are improved through the suspension stabilization and acid-resistant barrier functions of the shell. Furthermore, the surface charge of the shell is designed to be in the range of -20 mV to 0 mV, minimizing non-specific interactions with mucus and proteins while maintaining colloid stability even with changes in ionic strength within the gastric lumen. The dimensional design of the shell 2–8 nm and the complex 10–100 nm provides a balance point that allows it to spread widely through the gastric mucus network while minimizing adsorption and aggregation on the intestinal wall.

[0062] The iron oxide-based nanoparticle(s) in the orally administered complex according to one embodiment of the present invention are MRI T2 contrast agents that may reduce or nullify bright MRI signals originating from the fluids of the stomach and / or duodenum in T2-weighted images while remaining in the stomach. Oral administration of 5 mL to 200 mL of a solution containing the orally administered complex may stimulate the secretion of digestive fluids from the gallbladder, thereby enhancing the brightness of MRI signals in the bile ducts due to the digestive fluids in T2-weighted images. Additionally, the release of iron ions from the iron oxide-based particles in the highly acidic stomach may be inhibited by the polysaccharide-crosslinked colloidal particles coated on the core surface of the orally administered complex. Due to the polysaccharide-crosslinked colloidal particles coated on the core surface, the orally administered complex may exert a T2 contrast effect in the stomach for more than one hour.

[0063] In summary, the orally administered complex of the present invention stably exhibits a negative contrast effect that uniformly knurls gastric and duodenal water signals for an extended period after oral administration by (i) fixing and protecting the core surface with strongly bound anchor ligands via multi-point coordination, (ii) regulating the gastric acidic microenvironment and chelating free metal ions in situ with carboxylate / amine / hydroxyl residues, and (iii) inhibiting magnetization-induced aggregation and gravity-induced precipitation through weak negative charges and steric hindrance of cross-linked polysaccharides. This composition and mechanism of action are supported by results demonstrating no adverse effects in large animal MRCP efficacy, hepatic T2* stability, and repeated toxicity in beagles, and are universally applicable to various metal and metal oxide cores, thereby offering high clinical convertibility and platform scalability.

[0064]

[0065] [Polysaccharide Cross-linked Colloidal Particle-based Platform Technology]

[0066] The polysaccharide crosslinked colloidal particles of the present invention are characterized by being used as an oral stabilizing modifier that induces the formation of an acid-resistant barrier, inhibition of particle aggregation and inhibition of precipitation under gastric pH 1-2 conditions, by (i) modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, in an aqueous solvent with a first crosslinking agent having an epoxide group, and (a) forming a polysaccharide crosslinked particle (hydration average particle size 2-8 nm) by directly and / or intramolecularly and / or intermolecularly crosslinking between the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group, or (b) two spatially adjacent functional groups modified by the first crosslinking agent through a second crosslinking agent having two or more amine groups (-NH2), and (ii) controlling the surface charge to a range of -20 mV to 0 mV by modifying the number of basic amine groups derived from the crosslinking agent exposed on the surface to -COOH functional groups.

[0067] For example, crosslinking chemistry is implemented by a sequential reaction of epoxide (e.g., epichlorohydrin, diglycidyl ethers) and polyhydric amine (e.g., EDA, DETA, TETA), and surface -COOH introduction is carried out with organic acid anhydrides (e.g., succinic / maleic anhydrides).

[0068] The polysaccharide cross-linked colloidal particles of the present invention form a dense network through epoxide / polyamine double cross-linking and precisely control the zeta potential to -20 to 0 mV through partial carboxylation of surface -NH₂. These polysaccharide cross-linked colloidal particles function as a coordinate stacked shell for a single-additive stabilizer or a metal (oxide) core to form an acid-resistant barrier in gastric acid (pH 1-2) and prevent aggregation and precipitation by suppressing inter-particle attraction and bridging even under changes in magnetic field and ionic strength. As a result, the dispersibility of oral formulations is maintained for a long time, effective concentration and imaging signals are homogenized, and iron ion leaching and crystallinity degradation are suppressed, thereby improving T₂ imaging efficiency and inter-batch reproducibility. The polysaccharide cross-linked colloidal particle-based platform of the present invention is universally applicable to various polysaccharides, such as dextran, and various metal (oxide) cores.

[0069] The polysaccharide cross-linked colloid shell of the present invention simultaneously operates two physical and chemical mechanisms to prevent nanoparticles from sticking together and growing larger (bridge aggregation or aggregation / precipitation) even in environments with high acidity and high salt concentration, such as gastric juice. The first mechanism is "electrical repulsion by a weak negatively charged surface," and the second mechanism is a "steric barrier by cross-linked polysaccharide chains." These two mechanisms operate in combination to ultimately prevent the particles from approaching each other, even if attractive forces are generated between them.

[0070] Specifically, the details are as follows. The shell of the present invention adjusts the surface charge to between -20 mV and 0 mV by substituting a portion of the surface amine groups with carboxylic acid groups (-COOH / -COO-). At gastric pH 1–2, the carboxylic groups are partially protonated, so although the resulting charge is not a completely strong negative charge, a negative charge still remains on the surface, forming an electric double layer. As two nanoparticles approach each other, their respective electric double layers overlap, generating electrostatic repulsion. Although gastric juice has a high salt concentration, which shortens the thickness of the electric double layer (Debye length) and somewhat reduces the repulsion, the present invention is designed to maintain meaningful repulsion even at high ionic strengths by precisely controlling the surface charge density to -20–0 mV without excessively lowering it. This reduces the approach speed between particles and slows down the increase in the adjacent contact area.

[0071] At the same time, the polysaccharide cross-linked colloidal particle shell itself provides a thick and hydrated polymer layer. The polysaccharide chains form a strong hydration shell with water molecules, and when the polymer layers of two opposing particles attempt to overlap, an entropy penalty occurs due to osmotic pressure loss and chain compression. As a result, 'steric repulsion' arises, which dislikes the chains overlapping or being compressed. This steric repulsion operates regardless of the salt concentration of the solution and acts as a 'second safety mechanism' that remains unchanged even in situations where electrical repulsion is weakened due to high ionic strength. Furthermore, since the polysaccharide cross-linked colloidal particle shell of the present invention has a multivalent coordination bond structure, it forms a mechanically dense brush layer rather than a loose adsorption layer, thereby physically preventing direct contact between particle surfaces.

[0072] In the case of the present invention, the polysaccharide cross-linked colloid particles are already tightly fixed to the core surface metal ions through multi-point coordination bonds, so there is little room for the polymer chains to extend long toward the solution and attach to other particles. In addition, the carboxylates, hydroxyls, and residual amine groups remaining on the surface of the polysaccharide cross-linked colloid particles chelate metal ions in the solution.

[0073] Aggregation induced by dipole interactions due to magnetization is also weakened by the same principle. Even if the core is instantaneously magnetized under an external magnetic field, the weak negative charge on the surface and the hydrated polysaccharide cross-linked colloidal particle coating layer make it difficult for particles to align closely, thereby suppressing magnetization-induced aggregation. Consequently, even when adverse conditions such as the strong acidity of gastric juice, high salt concentration, and the strong magnetic field of an MRI scanner overlap, the complex of the present invention effectively prevents contact, aggregation, and precipitation between particles through the simultaneous operation of three mechanisms: electrical repulsion (electric double layer), steric repulsion (hydration layer), and bridge blocking (multi-point coordination fixation and metal ion chelation). Thanks to this stabilization, the complex maintains a uniformly dispersed state throughout the gastric cavity and continuously suppresses water signals without upper-lower concentration gradients over time, thereby producing a uniform negative contrast effect. This is clearly demonstrated, as confirmed in the examples, by the result that the knurling of the gastric and duodenal images remains evenly “darkened” for a long time after administration, and no heterogeneous contrast images in the form of precipitation or blotches appear.

[0074] The polysaccharide cross-linked colloid particles coated on the core surface of the acid-resistant nanocomposite of the present invention may be polysaccharide cross-linked colloid particles formed by cross-linking one to three linear polysaccharides or branched polysaccharides or two to thirty cyclic polysaccharides dispersed in an aqueous solvent with a cross-linking agent at the -OH functional groups of monosaccharides that are the building blocks thereof.

[0075] In polysaccharide cross-linked colloidal particles, the linear polysaccharide, branched polysaccharide, or cyclic polysaccharide to be cross-linked may be homopolysaccharides or heteropolysaccharides.

[0076] Non-limiting examples of linear polysaccharides, branched polysaccharides, or cyclic polysaccharides include dextran, cyclodextrin, maltodextrin, and inulin.

[0077] Inulin is an energy-storing polysaccharide found in plants of the Compositae family, consisting of linear chains of fructosyl groups connected by β-2,1 glycosidic bonds and terminated at the reducing end by an α-D-1,2 glucopyranoside ring.

[0078] Dextran is a polysaccharide derived from the condensation of glucose and is a complex branched glucan as shown in the structural formula below. It is a branched poly-α-d-glucoside of microbial origin that primarily has C-1 → C-6 glycosidic bonds. The main chain of the polymer consists of α(1→6) glycosidic bonds between glucose monomers, and the branched portions are connected by α(1→3) glycosidic bonds. In this specification, dextran also includes various derivatives thereof. Non-limiting examples of dextran derivatives include carboxymethyl dextran (CM dextran), dextran sulfate, and diethylaminoethyl dextran (DEAE-dextran). To provide polysaccharide cross-linked colloidal particles having a hydrated average particle size of 2 nm to 8 nm, the average molecular weight of the dextran or dextran derivative used in synthesis may be 10,000 Da or less, and the molecular weight of the spherical dextran cross-linked colloidal particles formed by cross-linking a dextran-based molecule through a cross-linking agent may be 90,000 Da or less. The polysaccharide cross-linked colloidal particles according to one embodiment of the present invention are nanoparticles formed in an aqueous solution by cross-linking a complex branched polysaccharide into an intramolecular and / or intermolecular group of a monosaccharide, which is a building block, through a cross-linking agent. The branched polysaccharide may form a dimer or trimer through intermolecular cross-linking, and the polysaccharide cross-linked colloidal particles may be formed by controlling only one branched polysaccharide molecule to undergo intramolecular cross-linking through a cross-linking agent. The degree of compression can be controlled by adjusting the degree of crosslinking and / or the molecular weight of the branched polysaccharide, and preferably, it can be a compact spherical shape.

[0079] The polysaccharide cross-linked colloidal particles of the present invention may be non-immunostimulating polysaccharide cross-linked colloidal particles that can be dispersed in aqueous media, such as lymph nodes, without gelation or aggregation, and satisfy pattern recognition receptor (PRR) and / or B cell receptor (BCR) non-operationality. Although the non-immunostimulating polysaccharide cross-linked colloidal particles of the present invention are formed by cross-linking linear polysaccharides or cyclic polysaccharides as well as complex branched polysaccharides with high immunogenicity, if 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are the building blocks of 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. The polysaccharide cross-linked colloid particles of the present invention can consistently achieve a non-immunostimulatory state by precisely defining the nanophysical properties (size, charge, and cross-linking substitution rate) and surface chemistry (epoxide selective modification → high-substitution cross-linking → surface -COOH post-modification) of the polysaccharide cross-linked colloids so as not to disrupt the innate-adapted immunity cross-pathway (PRR-Complement-BCR). The degree of immunogenicity of the polysaccharide cross-linked colloid particles of the present invention may vary depending on the degree of cross-linking, the size of the polysaccharide molecules, and specific chemical modifications.

[0080] Here, PRR / BCR non-operativity is defined in human PBMC or monocyte / macrophage-based in vitro tests as not showing an increase in TNF-α, IL-6, IL-1β secretion relative to the vehicle exceeding a pre-defined threshold, and / or signal amplification in TLR reporters being below the threshold, or C3a / C5a complement activation being within the acceptable range.

[0081] The particles of the present invention can be designed so that, under conditions where there is no targeting by cell surface ligands, the binding affinity with PRR or BCR is lower than a predetermined Kd upper limit (e.g., above the μM range) so that endocytosis does not significantly occur.

[0082] Antigens are foreign substances capable of triggering an immune response, and polysaccharides are easily targeted by innate and adaptive immunity due to their repetitive sugar sequences. Typically, when foreign substances enter the body, they are recognized by PRRs or BCRs, initiating a series of reactions such as cytokine secretion, complement activation, and antibody production; this can lead to the rapid elimination of drug carriers and side effects. In particular, since BCRs promote activation, differentiation, and antibody production when efficiently cross-linked by multivalent antigens, there is a risk of hypersensitivity reactions and the formation of memory B cells upon repeated administration. The crosslinking design of the polysaccharide crosslinked colloidal particles of the present invention lowers the degrees of freedom of the sugar chain and the accessibility of exposed carbohydrate epitopes through a high substitution rate of monosaccharide -OH (e.g., ≥ 60% of total monosaccharides, preferably ≥ 70%, more preferably ≥ 90% or more), thereby structurally reducing the BCR binding affinity and crosslinking potential established by a complex of hydrogen bonding, van der Waals, and hydrophobic interactions. As a result, the polysaccharide crosslinked colloidal particles of the present invention are difficult to recognize as multivalent antigens, and thus the potential for BCR clustering and antibody induction is significantly low.

[0083] The polysaccharide cross-linked colloidal particles of the present invention can form a compact spherical shape by modifying 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are the building blocks of the polysaccharides, by a cross-linking agent, and can also efficiently reduce or minimize immunogenicity to immune cells relative to the polysaccharides to be cross-linked. In addition, not only are they not hydrolyzed by enzymes in the body, but they can also minimize immunogenicity caused by the site exposed by hydrolysis.

[0084] At this time, the polysaccharide crosslinked colloidal particles of the present invention, in which the -OH of the polysaccharide dispersed in an aqueous solvent is modified by a crosslinking agent, preferably, at least one -OH of at least one monosaccharide among two consecutive monosaccharides in the polysaccharide is mostly modified by a crosslinking agent, thereby not being hydrolyzed by enzymes in the body and reducing or minimizing immunogenicity against immune cells.

[0085] The polysaccharide crosslinked colloidal particles of the present invention are water-soluble colloidal amorphous nanoparticles in which linear polysaccharides (e.g., inulin) dispersed in an aqueous solvent, as well as complex branched polysaccharides (e.g., dextran) or cyclic polysaccharides (e.g., cyclodextrin) dispersed in an aqueous solvent, are crosslinked intramolecularly and / or intermolecularly to form a more complex three-dimensional network, or as described above, at least 60%, at least 70%, at least 90%, or at least 95% of the total number of monosaccharides, which are the building blocks of polysaccharides dispersed in an aqueous solvent, are modified by the crosslinking agent, or at least one -OH functional group in at least one of two consecutive monosaccharides in the polysaccharide chain is mostly modified by the crosslinking agent, thereby minimizing immunogenicity to immune cells without being hydrolyzed by enzymes in the body.

[0086] Non-limiting examples of enzymes in the body include glycoside hydrolases, which hydrolyze the glycosidic bonds of polysaccharides into simple sugars.

[0087] Glycoside hydrolases are found in almost all regions of living organisms. In intestinal prokaryotes, they are found as intracellular and extracellular enzymes primarily involved in nutrient acquisition. In bacteria, one of the important occurrences of glycoside hydrolases is the enzyme beta-galactosidase (LacZ), which is involved in regulating the expression of the lac operon in E. coli. In higher organisms, glycoside hydrolases are found in the endoplasmic reticulum and Golgi apparatus as enzymes involved in the processing of N-linked glycoproteins, and in lysosomes as enzymes involved in the breakdown of carbohydrate structures. Glycoside hydrolases are found in the intestine and saliva and break down complex carbohydrates such as lactose, starch, sucrose, and trehalose. In the intestine, they are found in endothelial cells in the form of glycosylphosphatidyl fixation enzymes. Glycoside hydrolase is involved in the biosynthesis and breakdown of glycogen in the body.

[0088] The polysaccharide cross-linked colloidal particles of the present invention may be provided by a manufacturing method comprising the following steps as a non-limiting example:

[0089] Step 1: preparing an aqueous solution of linear polysaccharides, branched polysaccharides, or cyclic polysaccharides;

[0090] A second step of modifying the -OH functional groups of monosaccharides, which are the building blocks of polysaccharides, with the first crosslinking agent by adding a first crosslinking agent having an epoxide functional group that reacts with the hydroxyl group (-OH) of the polysaccharide with an alkaline aqueous solution;

[0091] A third step of dropwise adding a second crosslinking agent having two or more amine groups (-NH2) to produce polysaccharide crosslinked colloid particles having terminal amine groups derived from the second crosslinking agent on the surface, wherein spatially adjacent functional groups modified by the first crosslinking agent are intramolecularly and / or intermolecularly crosslinked through the second crosslinking agent;

[0092] A fourth step of administering an organic acid anhydride to polysaccharide crosslinked colloid particles having terminal amine groups derived from a second crosslinking agent on their surface to modify some or all of the terminal amine groups into carboxylic acid groups and / or carboxylate groups; and

[0093] Step 5, optionally, by administering an aqueous solution of metal oxide (e.g., iron oxide) nanoparticles to the water-dispersible polysaccharide cross-linked colloidal particles prepared in the previous step, to prepare a composite in which the surface of the metal or metal oxide nanoparticles is modified into polysaccharide cross-linked colloidal particles.

[0094] In order to prevent an 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 relative to the polysaccharide being crosslinked without being hydrolyzed by enzymes in the body so as to be excreted without exposure to potential sugar-based immunogenic sites, the present invention crosslinks linear polysaccharides, branched polysaccharides, or cyclic polysaccharides intramolecularly and / or intermolecularly in an aqueous solution.

[0095] At this time, it was discovered that when branched polysaccharides such as dextran are crosslinked by a crosslinking agent, 2 to 3 branched polysaccharide molecules form a spherical core through intramolecular and intermolecular crosslinking. Similarly, it was possible to provide hydration-sized polysaccharide crosslinked colloid particles that do not penetrate the blood vessel walls of normal capillaries but are filtered in the renal capillaries through intramolecular and intermolecular crosslinking, with 2 to 3 for linear polysaccharides and up to 30 for cyclic polysaccharides.

[0096] The above polysaccharide crosslinked colloidal particles are spherical amorphous polysaccharide crosslinked colloidal particles formed by (a) direct between the functional group modified by the first crosslinking agent and the spatially adjacent functional group modified by the first crosslinking agent and / or (b) intramolecular and / or intermolecular crosslinking between two spatially adjacent functional groups modified by the first crosslinking agent through a second crosslinking agent having two or more amine groups (-NH2).

[0097] Polysaccharide cross-linked colloidal particles can control the number of linear polysaccharide, branched polysaccharide, or cyclic polysaccharide molecules to be cross-linked to be the same or different through their synthesis conditions and / or purification.

[0098] For example, the present invention can produce polysaccharide crosslinked colloid 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 polysaccharides are modified by the crosslinking agent at the -OH functional groups of monosaccharides that are building blocks of polysaccharides, by adding and reacting (i) a first crosslinking agent having an epoxide group that reacts with the hydroxyl group (-OH) of the polysaccharide and a functional group that chemically bonds with the hydroxyl group (-OH) and / or amine group (-NH2) and (ii) a second crosslinking agent having two or more amine groups (-NH2) to an aqueous solution of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide through the second and third steps, thereby modifying and crosslinking the monosaccharides that are building blocks of the linear polysaccharide, branched polysaccharide, or cyclic polysaccharide with the crosslinking agent.

[0099] In the second step, the first crosslinking 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 the -OH functional group site of the monosaccharide is modified so that (a) between the functional group modified by the first crosslinking agent and the spatially adjacent -OH functional group, and (b) two spatially adjacent functional groups modified by the first crosslinking agent can react with the second crosslinking agent, and preferably, it may be a halo alkyl oxirane, for example, epichlorohydrin.

[0100] In the third step, the second crosslinking agent having two or more amine groups (-NH2) can be replaced with any crosslinking agent capable of covalently bonding to the functional group derived from the first crosslinking agent that modifies the -OH functional group site of the monosaccharide, and this also falls within the scope of the present invention.

[0101] In one embodiment of the present invention, when epichlorohydrin is used as a first crosslinking agent to modify the -OH functional groups of monosaccharides, ethylenediamine or diethylenetriamine (DETA) may be used as a second crosslinking agent to participate in crosslinking between spatially adjacent modified functional groups.

[0102] When linear polysaccharides, branched polysaccharides, or cyclic polysaccharides react with epichlorohydrin as a first crosslinking agent and ethylenediamine or DETA as a second crosslinking agent, a series of chemical modifications occur that significantly change the structure and properties of the polysaccharides.

[0103] According to the present invention, when polysaccharides are modified (combined modifications) by using epichlorohydrin as a first crosslinking agent and ethylenediamine or DETA as a second crosslinking agent, the crosslinking increases and stability is improved.

[0104] In particular, terminal amine groups derived from the second crosslinking agent exposed on the surface of polysaccharide crosslinked colloid particles can impart new properties such as enhanced chelating ability, increased reactivity, and additional functionalization potential. Therefore, polysaccharide crosslinked colloid particles having terminal amine groups derived from the second crosslinking agent on their surface, generated in the third step, can enhance their ability to interact with other molecules, such as chelation or drug binding, through the amine groups (-NH2).

[0105] Polysaccharide crosslinked colloidal particles having terminal amine groups derived from the second crosslinking agent formed in the third step 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 -20 mV to 0 mV and enabling the particle to have a biocompatible pH. Additionally, the number of amine groups derived from the crosslinking agent exposed on the surface of the polysaccharide crosslinked colloidal particles of the present invention can be controlled through a reaction to introduce -COOH-containing functional groups.

[0106] For example, when amine groups derived from the crosslinking agent exposed on the surface of polysaccharide crosslinked colloid particles are modified into -COOH-containing functional groups, the surface charge can be freely controlled within the range of -20 mV to 0 mV by adjusting the type and / or degree of modification of the -COOH-containing functional groups. In the fourth step, for example, by adjusting the amount of succinyl anhydride (SA), the surface charge can be freely controlled within the range of -20 mV to 0 mV. As the amount of added SA increases, the surface charge becomes negative.

[0107] In the polysaccharide crosslinked colloid particles of the present invention, the interaction with water and other molecules is altered when the amine group derived from the crosslinking agent exposed on the surface is replaced with a carboxyl group.

[0108] For example, in the fourth step, the amount of organic acid anhydride administered can be controlled to control the number of amine groups derived from the crosslinking agent exposed on the surface of the polysaccharide crosslinked particles of the present invention. Accordingly, by controlling the reaction ratio with the drug, the number of functional molecules (e.g., drugs, targeting molecules) bound to the polysaccharide crosslinked colloid particles can also be controlled.

[0109] The polysaccharide crosslinked colloidal particles of the present invention can be coordinately bonded to a core composed of iron oxide-based nanoparticle(s) through crosslinking agent-derived amine groups and / or -COOH functional groups exposed on the surface thereof.

[0110] The polysaccharide crosslinked colloidal particles of the present invention enable strong coordination bonding between a crosslinking agent-derived functional group (carboxyl group or amine group) and a core surface composed of metal or metal oxide-based nanoparticle(s), thereby imparting colloidal stability.

[0111] In step 5, non-limiting examples of hydrophilic functional groups that coordinate with the core surface composed of metal or metal oxide-based nanoparticle(s) include hydroxy, carboxylic acid, carboxylate, amine, etc.

[0112] The polysaccharide cross-linked colloid particles according to the present invention can be designed and synthesized so that the contrast effect is maintained for a relatively long time by the surface-modified orally administered complex with polysaccharide cross-linked colloid particles, thereby extending the scan time during MRI imaging and improving the spatial resolution of MRI, thus enabling imaging at a higher resolution.

[0113] In addition, a core composed of metal or metal oxide-based nanoparticle(s), such as iron oxide, designed to act as a T1 or T2 MRI contrast agent, is surface-modified into polysaccharide cross-linked colloidal particles in the fifth step, so that it can move between internal structures without aggregation upon injection into the body and thus perform its role as a T1 or T2 MRI contrast agent.

[0114] Meanwhile, according to the present invention, polysaccharide crosslinked colloid particles formed by intramolecularly and / or intermolecularly crosslinking linear polysaccharides, branched polysaccharides, or cyclic polysaccharides with a crosslinking agent may be subjected to chemical modifications such as adding, removing, or modifying functional groups (-COOH, -NH2, -OH, etc.) so that they are exposed on the surface. Such modifications may be performed to impart new chemical properties or to enhance existing properties.

[0115] Polysaccharide cross-linked colloidal particles can optimize physical properties, such as solubility or viscosity, for specific applications by controlling the type of linear, branched, or cyclic polysaccharides and / or their molecular weight and / or cross-linking density. Furthermore, chemical modifications can be aimed at introducing new functionalities or enhancing specific chemical interactions.

[0116] The 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 during cross-linking, the amount and administration rate of the cross-linking agent administered during the synthesis reaction, and additional chemical functional group modification, and can finally impart desired blood circulation time and desired pharmacokinetics of distribution and elimination in the body.

[0117] In the present invention, the crosslinking agent-derived functional group exposed on the surface of the polysaccharide crosslinked colloidal particle may be the terminal functional group of the crosslinking agent itself or a modified / substituted functional group thereof. For example, the crosslinking agent-derived functional group exposed on the surface of the polysaccharide crosslinked colloidal particle may be one in which at least some of the functional groups of the crosslinking agent exposed on the surface have been modified / substituted.

[0118] In the present invention, the hydrophilic functional group may be derived from a functional group of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide that did not participate in the crosslinking reaction, a functional group of a crosslinking agent that did not participate in the crosslinking reaction, and / or a functional group obtained by further modifying a one-terminal of a crosslinking agent exposed after the crosslinking reaction.

[0119] Non-limiting examples of functional groups derived from the crosslinking agent or hydrophilic functional groups exposed on the surface of polysaccharide crosslinked colloid particles include amine groups, carboxyl groups, hydroxyl groups, and / or thiol groups. Reactive functional groups such as amines, thiols, carboxyl groups, and hydroxyl groups facilitate not only surface modification but also chemical binding with biopharmaceuticals or various types of small molecule drugs, such as ligands that specifically bind to receptors on specific cells, antibodies or fragments thereof, antigenic peptides, and nucleic acids (DNA, RNA, or fragments thereof). Additionally, in some cases, the active form of the drug may be released by degradation in the acidic atmosphere (pH ≤ 7) surrounding the cancer or by hydrolytic enzymes. For example, non-limiting examples of acid-sensitive bonds that degrade in the acidic atmosphere (pH ≤ 7) surrounding the cancer include carbonate or ester bonds.

[0120] If the functional groups exposed on the surface of the polysaccharide cross-linked colloid particles of the present invention carry a positive charge such as amines, cytotoxicity may occur as with other cationic polymers, but this can be resolved by substituting some or all of the amine groups with carboxyl groups, methyl groups, ethyl groups, etc.

[0121] Non-limiting examples of hydrophilic functional groups that coordinate with the iron of iron oxide include amines, thiols, carboxyls (carboxylates and carboxylic acids), and hydroxyls.

[0122] Accordingly, the polysaccharide cross-linked colloidal particles of the present invention can be used to produce various polysaccharide cross-linked colloidal particle derivatives with unique properties suitable for specific applications through chemical modifications of functional groups such as -COOH, -NH2, and -OH exposed on their surface, and these also fall within the scope of the present invention.

[0123]

[0124] [Stimulation of digestive fluid secretion and gastrointestinal transit time]

[0125] When the oral solution containing the complex of the present invention is administered orally, it acts as a T2-negative contrast agent during its retention in the gastric cavity, temporarily lowering gastric and duodenal water signals, while simultaneously inducing gallbladder contraction and bile (digestive fluid) secretion as physiological responses. Specifically, after orally administering 5 mL to 200 mL of the complex-containing solution, it was confirmed that the signal of the bile duct column in T2-weighted images became relatively brighter compared to before administration. This is interpreted as a result of increased bile duct fullness due to an increase in the amount of bile secreted from the gallbladder. This contrast amplification occurs in parallel with the nulling of the water signal in the gastric cavity and improves the visibility of the distal end of the bile duct and the adjacent section of the duodenal papilla.

[0126] The above-mentioned increase in bile secretion is presumed to be the result of induced gallbladder contraction caused by the activation of the cholecystokinin (CCK) pathway due to gastric wall elongation and duodenal afferent stimulation resulting from the retention of the complex-containing solution in the gastric cavity, as well as stimulation of intestinal mucosal receptors in response to changes in osmotic pressure and acidity. The formulation of the present invention can be designed with a pH of 3.0 to 8, an osmotic pressure of 270 to 330 mOsm / kg, and a viscosity of 1 to 80 mPa·s, thereby enabling a balance between appropriate retention and the induction of physiological responses while minimizing rapid stimulation in the gastric cavity.

[0127] Figure 13 shows representative time-lapse images in a mouse model, in which the water signal in the gastric cavity is observed to be high before administration (pre-injection), but in images acquired at 10, 30, and 60 minutes after administration (post-injection), the signal consistently degrades across the entire gastric cavity boundary, demonstrating that knurling is maintained. This is possible because the polysaccharide cross-linked colloid shell of the present invention acts as a 'strongly bound anchor ligand' and an 'acid-resistant barrier layer' even in gastric fluid (pH approx. 1-2), preserving the magnetism of the core and achieving 'aggregation inhibition (colloid stabilization)' and 'precipitation inhibition (suspension stabilization).' Even under exposure to an external magnetic field, magnetization-induced aggregation is inhibited by the shell's surface charge (-20 mV to 0 mV) and steric hindrance, ensuring that the contrast agent is uniformly distributed throughout the gastric cavity and maintaining image quality without layer separation over time (see Example 11-1 and Figure 13).

[0128] In addition, in the composite of the present invention, polysaccharide cross-linked colloidal particles with a hydration average particle size of 2 nm to 8 nm coating the core surface are not only firmly fixed to the surface by coordinating with iron ions of the core, but also simultaneously present hydroxyl groups (-OH), amine groups (-NH2) derived from the crosslinking agent, and carboxyl groups (-COOH) exposed on the surface without coordination. These functional groups inhibit the diffusion into the solution phase by chelating or coordinating free iron ions that may be generated in trace amounts under gastric acid conditions on the surface, thereby reducing redox chain reactions and metal ion-mediated aggregation. With the combination of these microenvironmental control effects and barrier layer effects, the T2 contrast effect is stably maintained in the gastrointestinal tract for at least 1 hour.

[0129] Since gastrointestinal transit time varies by species and dietary status, it is advisable to set the image acquisition time based on the physiological characteristics of the target animal or human. Generally, mice exhibit rapid gastric emptying due to their high metabolic rate and small body size, with the gastric emptying half-life of liquid contents reported to be approximately one hour. As medium-sized mammals, pigs show gastric emptying characteristics that are slower than mice but faster than humans; furthermore, their digestive physiology and anatomy are similar to humans, making them suitable as preclinical models for oral formulations. In humans, the gastric emptying half-life of liquids ranges from approximately tens of minutes to one hour, while solid foods take several hours; therefore, when applying MRCP clinically, it is advantageous to select the optimal time within the range of 10 to 120 minutes after administration, tailored to the equipment and patient condition. This time design can be adjusted so that the period during which the knurling effect is maximized during gastric retention sufficiently overlaps with the period during which the bile duct contrast becomes relatively brighter due to gallbladder contraction and bile secretion.

[0130] In summary, the orally administered complex of the present invention acts as a T2-negative contrast agent while remaining in the stomach and duodenum, stably nulling the water signal of the gastric cavity and simultaneously increasing the relative signal of the bile duct by stimulating bile secretion following gallbladder contraction. This improves the visibility of the bile duct and pancreatic duct in T2-weighted images, thereby allowing for more accurate identification of causative lesions of biliary-pancreatic duct diseases such as cancer, inflammation, gallstones, pancreatic stones, bile duct stenosis, or pancreatic duct stenosis.

[0131]

[0132] [MRI contrast agent pharmaceutical composition and biliary and pancreatic duct imaging diagnostic composition]

[0133] The present invention provides a pharmaceutical composition for MRI contrast agents and a composition for diagnosing biliary and pancreatic duct imaging, comprising the aforementioned orally administered core-shell complex as an active ingredient. The complex consists of a metal or metal oxide core (preferably Fe3O4 or γ-Fe2O3) and a shell coated with polysaccharide cross-linked colloidal particles controlled to have a hydration average particle size of 2 to 8 nm and a surface charge of -20 mV to 0 mV. The shell forms multi-point coordination bonds with metal ions on the core surface to function as a strongly bound anchor ligand in gastric fluid, provides an acid-resistant barrier layer, and inhibits aggregation and precipitation by controlling surface charge, steric hindrance, and composition viscosity. As a result, the hydration average particle size of the entire complex is maintained in the range of 10 to 100 nm, metal ion leaching is minimized even in the gastric lumen (pH 1 to 2), and r2 attenuation efficiency is stably preserved.

[0134] A pharmaceutical composition is prepared by dispersing the complex in an orally acceptable carrier. The carrier is based on purified water and can be adjusted to have a pH of 3.0 to 8 and an osmotic pressure of 270 to 330 mOsm / kg. The formulation may be provided as an oral suspension or a powder / concentrate for reconstitution. If necessary, it may include a viscosity modifier (e.g., xanthan gum 0.05 to 0.3%) to impart viscosity for improved suspension stability, a buffer salt or chelating agent (e.g., citrate 1 to 10 mM) for chemical stability and microenvironment buffering, or a preservative.

[0135] A composition for diagnosing biliary and pancreatic duct imaging contains the above complex as a contrast agent and is used to selectively reduce gastric and duodenal fluid signals in MRCP to increase the relative contrast of the bile ducts and pancreatic ducts. As an example of administration, a solution containing the complex is orally administered at a dose of 0.05 to 5 mL per kg of body weight (corresponding to 0.05 to 1 mg / kg of iron) 10 to 120 minutes before image acquisition. Fasting is adjusted according to the clinical situation, and the timing of imaging is set considering the gastric residence time. Suitable sequences for use are T2-weighted single / multi-shot turbo spin-echo (HASTE, RARE, FRFSE, etc.) and are operated on 1.5 to 3.0 T clinical equipment.

[0136] After oral administration, the complex remains in the gastric lumen and, if necessary, the duodenum, undergoing homogeneous dispersion. The superparamagnetic core induces local magnetic sensitivity differences, increasing the spin phase desynchronization of hydrogen protons and shortening T2 (or T2*) time. This process is maintained stably for at least one hour due to the shell's acid-resistant barrier and colloidal stabilization, uniformly degrading water signals throughout the gastrointestinal tract and causing them to appear null in the image. Magnetization-induced aggregation is suppressed by the shell's surface charge and steric hindrance, preventing delamination and localized overconcentration, which is confirmed by the spatial homogeneity of signal degradation. Additionally, physiological responses to oral administration, such as increased gallbladder contraction and bile secretion, lead to higher fluid column filling in the bile ducts. This results in bile duct high signals appearing relatively brighter in T2-weighted images, further amplifying contrast.

[0137] Clinically, this composition improves anatomical continuity and lesion contrast in areas that were difficult to observe in conventional MRCP due to the superposition of high signals from the stomach and duodenum, such as the distal part of the common bile duct, the section adjacent to the duodenal papilla, and the terminal part of the pancreatic duct. It also allows for the identification of pathological findings such as defect signals caused by gallstones / pancreatic stones, changes in the diameter of the stenotic area and proximal dilation, inflammatory wall thickening, and microsludge with high contrast, thereby contributing to clinical decision-making, such as the differentiation of calculous, inflammatory, and malignant stenosis and the determination of indications for ERCP.

[0138] In T2-weighted MRCP, gastric and duodenal fluid signals are selectively reduced after administration, significantly increasing the relative contrast of the bile ducts and pancreatic ducts. Consequently, pathological findings such as high-signal defects of bile duct stones (cholesterol stones and pigment stones), changes in lumen diameter and proximal dilation at bile duct and pancreatic duct stenosis sites, cholestasis and gallbladder distension, and irregular dilation or protruding lesions of the pancreatic duct are observed more clearly than before administration. In particular, the visibility of lesions in the distal common bile duct, the section adjacent to the duodenal papilla, and the terminal pancreatic duct—which were difficult to evaluate in conventional MRCP due to the superposition of gastric and duodenal high signals—is improved, allowing for more accurate identification of the presence or absence of cancer, inflammation, gallstones, pancreatic stones, bile duct stenosis, or pancreatic duct stenosis. This composition for biliary and pancreatic duct imaging acts as a T2 contrast agent while remaining in the stomach and / or duodenum to reduce gastrointestinal signals, while simultaneously stimulating gallbladder contraction and bile secretion as physiological responses. A transient increase in bile flow increases the filling of the bile duct fluid column, making the bile duct high signal relatively brighter in T2-weighted images and further amplifying contrast. This formulation-physiological integration effect enhances the detection of microscopic findings such as micronodules, calculi or sludge, and mild stenosis-dilation patterns within the bile duct, and clinically contributes directly to determining treatment strategies, including the differentiation of calculous, inflammatory, and malignant stenosis, assessment of ERCP indications, and preoperative planning.

[0139] In terms of safety, this oral composition exhibits a profile centered on local gastrointestinal action due to its acid-resistant barrier layer and colloid / suspension stabilization, thereby reducing the potential for accumulation in the liver and spleen following systemic absorption. In preclinical single high-dose studies, no significant toxicity was observed in the range of hundreds to thousands of times the clinically planned dose, and no accumulation trend was observed through liver T2 time-course analysis in large animal models. The formulation is designed considering preservation, redispersibility, equipment compatibility (plastic / glass containers), and patient compliance (taste and ease of swallowing), and quality specifications include pH, osmotic pressure, viscosity, microbial limits, metal impurities, r2 and magnetic retention rates, free metal ion release upon 120 minutes of exposure to artificial gastric fluid (less than 5% of total metals), and the absence of precipitation.

[0140] The acid-resistant nanocomposite of the present invention is based on a core-shell structure comprising a metal (or metal oxide) core and a shell coated with polysaccharide cross-linked colloidal particles (hydrated 2–8 nm, surface charge -20–0 mV). The shell forms multi-point coordination bonds with the core metal ions to suppress dissolution and crystallinity damage in gastric acid, thereby preserving superparamagnetism and r2 efficiency. The surface charge and cross-linking network blocks magnetization-induced aggregation and precipitation, maintaining homogeneous T2-negative contrast (signal cancellation) during gastric lumen retention. Increased bile secretion due to gallbladder contraction leads to greater relative contrast, improving the continuity of the bile and pancreatic ducts and the visibility of lesions in MRCP. The total particle size of 10–100 nm and the weakly charged surface reduce mucosal absorption and systemic exposure, thereby increasing safety, and the ease of process control ensures excellent batch-to-batch reproducibility.

[0141] Figure 1a illustrates the limitations of conventional MRCP. Through the anatomical structure of the stomach and duodenum, and the bile and pancreatic ducts (left), an MRCP schematic (middle), and an actual MRCP image (right), it illustrates a situation where strong T₂ signals from the stomach and duodenum overlap with signals from the bile and pancreatic ducts, making it difficult to observe the distal bile duct.

[0142] Figure 1b schematically illustrates a strategy to selectively image the bile ducts and pancreatic ducts in high contrast by removing disturbance signals occurring in the stomach / duodenum using a negative contrast agent.

[0143] Figure 1c illustrates the technical limitations of existing iron oxide-based oral T2 contrast agents. (1) the occurrence of aggregation and precipitation and reduced contrast effect due to low colloidal stability caused by unstable coating, and (2) the problem of reduced stability caused by the easy decomposition of iron oxide in the highly acidic / high-ionic environment of gastric fluid, are shown in the diagram and schematic (dispersion-aggregation curve, conceptual diagram of decomposition in gastric fluid).

[0144] Figure 2a is a conceptual diagram of the structure of dextran cross-linked colloidal particles. It illustrates a platform technology based on dextran cross-linked colloidal particles in which the -OH group of the dextran chain forms a multi-point bondable functional group (e.g., -COOH) on the surface of spherical particles formed by an epoxide / polyamine cross-linking reaction, thereby enabling strong coordination bonding to the surface of iron oxide.

[0145] Figure 2b summarizes the mechanism of action and characteristics of a shell-structured composite consisting of an iron oxide particle core coated with dextran-crosslinked colloid particles. It indicates that the iron oxide particle core is protected even in acidic conditions (low pH) by multi-point coordination bonding between the dextran-crosslinked colloid particles and the iron oxide particles, and that colloid / pH stability is maximized by inhibiting gastric aggregation through the formation of a hydration layer by the dextran-crosslinked colloid particle coating. On the right, a 7 nm Fe3O4 core (T2 relaxivity r2 ≈ 217 mM -1 ·s -1 It demonstrated linearity dependent on T2 concentration and superior contrast effect per unit concentration compared to existing formulations.

[0146] FIG. 2c is a conceptual diagram of an acid-resistant nanocomposite with a multicore (bridged) core-shell structure. For example, multiple iron oxide cores (e.g., Fe3O4, approx. 8 ± 1 nm) are covalently cross-linked to each other by a shell composed of dextran cross-linked colloidal particles (hydration average 2–8 nm, surface charge -20–0 mV) and function as a single particle. This indicates that some colloidal particles form multi-point coordination bonds with two or more core surface metal ions to bridge adjacent cores. The hydration diameter of approx. 45 nm observed in Example 2 and FIG. 6 corresponds to a value considering the hydration layer, etc., for a core thickness of 8 nm × 3 + shell thickness of 4 nm × 4 (≈40 nm), thereby supporting a covalently cross-linked structure between cores. This structure provides an acid-resistant barrier that protects the core in gastric acid (pH 1-2) and colloid stabilization simultaneously, thereby inhibiting aggregation and precipitation and enabling uniform T2-negative contrast imaging in MRCP.

[0147] Figure 3 is a graph showing the TEM images of INV-003 (left, middle) and the size measured from the TEM images (right).

[0148] Figure 4 shows the XRD analysis results of INV-003 and a comparison with JCPDS.

[0149] Figure 5 shows the IR spectrum (a) and peak assignment (b) of INV-003.

[0150] Figure 6 is a graph showing the hydration diameter of INV-003.

[0151] FIG. 7a is a conceptual diagram of the colloidal stability of a mononuclear core-shell structure or a multinuclear (bridged) core-shell structure behaving as a single hydrated particle in a dynamic fluid environment according to one embodiment of the present invention. It indicates that the dispersed state is maintained even in a high concentration salt and biological pH (5-7) environment.

[0152] Figure 7b shows a photograph of a glass vial observed for 7 days under pH 5 / 7 / 9 and NaCl 0-1000 mM conditions, showing that no precipitation due to aggregation was observed for INV-003 (precipitation for the control iron oxide).

[0153] Figure 7c shows the change in hydration diameter over time as a function of DLS at pH 5 / 7 / 9 (top) and NaCl 250 / 500 / 1000 mM (bottom), confirming high colloidal stability with no increase in size over 7 days.

[0154] Figure 8 is the magnetic hysteresis curve of INV-003.

[0155] Figure 9 shows the measurement results of the relaxivity coefficient of INV-003. (a) T2 image of the INV-003 phantom. (b) Graph showing 1 / T2 values ​​according to the concentration of INV-003.

[0156] Figure 10 shows the change in hydration diameter of INV-003 according to pH (HCl concentration) (a), the change in T2 relaxation time (c), and the change in signal observed in the T2 image (b).

[0157] Figure 11 shows clinical symptoms (a) and body weight changes (b) following oral administration of INV-003.

[0158] Figure 12 shows clinical symptoms (a) and body weight changes (b) following intravenous administration of INV-003.

[0159] Figure 13 shows the results of a magnetic resonance cholangiopancreatography efficacy test showing a reduction (dark contrast) or nulling of the T2 MRI signal in the stomach following oral administration of INV-003 to mice.

[0160] Figure 14a is a representative magnetic resonance cholangiopancreatography (MRCP) image performed after oral administration of INV-003 to pigs, showing that gastric / duodenal signals are canceled out and the continuous pathways of the bile duct and pancreatic duct are clearly visualized in high contrast (after administration).

[0161] Figure 14b shows a comparison of pre- and post-administration images in the same animal, showing that before administration, gastric signals were high and the bile duct and pancreatic duct pathways were unclear, whereas after administration of INV-003, gastric and duodenal signals decreased, selectively highlighting the anatomical pathways of the bile duct and pancreatic duct (Left: before administration, Right: after administration).

[0162] Figure 14c shows the results of quantitative analysis in three pigs, indicating that at different dosages (0.063, 0.125, 0.250 mg Fe / kg), the gastric contrast-to-noise ratio (CNR) after administration was reduced by more than 10 times compared to before administration, showing that gastric signals were completely eliminated.

[0163] Figure 15a shows changes in body weight in a single-dose toxicity test in rodents. After single-dose administration of doses up to 480, 2,000, and 1,280 times the clinically planned dose to ICR mice (intravenous, oral) and SD rats (oral), body weight increased normally for 14 days, indicating no signs of acute toxicity.

[0164] Figure 15b shows the results of comparing hepatic T2* relaxation times in pigs before and immediately after oral administration of INV-003, and on days 7 and 13 after administration. The mean values ​​at all time points were similar, suggesting that hepatic iron accumulation or systemic absorption was not significant.

[0165] Figure 16 is a schematic diagram of the design of an optimal dose search study to determine the optimal concentration and dosage of the oral negative contrast agent INV-003 in healthy pigs. Formulation concentrations of 0.05–0.40 mg / mL and dosages of 0.063, 0.125, and 0.250 mg Fe / kg were screened stepwise, and the sample size (N) for each step was indicated.

[0166] Figure 17 illustrates representative MRCP images before (Pre) and after (Post) oral administration of formulations at concentrations of 0.05, 0.10, 0.20, and 0.40 mg Fe / mL. It shows that after administration, gastric / duodenal signals are eliminated, selectively highlighting the contours and continuity of the bile ducts and pancreatic ducts.

[0167] Figure 18 is a bar graph showing the SNR_pre·SNR_post and CNR_pre·CNR_post values ​​calculated by group in an adaptive dose exploration study. After administration of INV-003 (Post), gastrointestinal SNR and CNR consistently decreased significantly, suggesting a gastrointestinal signal suppression effect.

[0168] Figure 19 is a figure comparing the pre- and post-administration changes in SNR and CNR for three dosages of 0.25, 0.125, and 0.063 mg Fe / kg under a concentration of 0.4 mg / mL in the reproducibility evaluation. The reproducibility of the effect was confirmed by presenting similar signal reduction patterns and standard deviation ranges for each dosage.

[0169] Figure 20 is a diagram comparing the liver T2* map (top) and T2* relaxation time (bottom) before administration of INV-003 (0 hours) and at 7 and 13 days after administration in vivo. The difference in average T2* values ​​between time points is not large, showing that changes in systemic absorption or iron accumulation are minimal.

[0170] The present invention will be explained in more detail below 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.

[0171] In the examples below, the analysis of most physical properties of the complex referred to as INV-003, including synthesis, structure, morphology, magnetism, and MRI efficacy analysis, has been completed.

[0172] Example 1: Synthesis of Dextran T-10 Based Polysaccharide Cross-linked Colloidal Particles

[0173] 180 μmol of dextran T-10 (average molecular weight 10,000 Da) was dissolved in 9 mL of distilled water, and then 75 mmol of epichlorohydrin and 75 mmol of NaOH were added. 380 mmol of ethylenediamine was added to this solution and stirred at room temperature (RT) for 24 hours. 25 mg of succinic anhydride was added to this solution, and after 24 hours of succinylation, the solution was purified using a 10 kDa molecular weight cutoff (MWCO) filter. The hydrodynamic size measured by DLS is 5 nm.

[0174] Example 2: Synthesis of core iron oxide nanoparticles and surface modification by polysaccharide cross-linked colloidal particles

[0175] 0.125 mL of a 0.24 M iron(III) chloride hexahydrate solution and 0.125 mL of a 0.12 M iron(II) chloride tetrahydrate solution were mixed. Then, a 1 M tetramethylammonium hydroxide (TMAOH) solution was slowly added dropwise, followed by sonication for 3 hours. The polysaccharide cross-linked colloidal particle solution of Example 1 was added to this solution, and the mixture was stirred at room temperature for 12 hours to induce a coordination bond coating on the surface of the iron oxide particles (core). Subsequently, the solution was washed and concentrated using a 100 kDa molecular weight cutoff filter to obtain the INV-003 colloidal solution. Under TEM, the core crystal size was 8.0 ± 1.0 nm (Fig. 3), and the core-shell complex DLS hydration diameter was 47.1 ± 4.3 nm (Fig. 6).

[0176] Example 1 specifically implemented intramolecular / intermolecular crosslinking of polysaccharides and the introduction of surface -COOH using an epoxide first crosslinking agent and a polyhydric amine second crosslinking agent in an aqueous solvent to produce polysaccharide crosslinked colloidal particles of several nm size. Example 2 completed a stable core-shell complex (INV-003) of the 10–100 nm range by synthesizing an iron oxide core and then forming a coordinate bond coating with metal ions on the core surface through functional groups that form multi-point coordinate bonds on the shell coated with the polysaccharide crosslinked colloidal particles. The core crystal size (approx. 8 nm) in TEM and the complex hydration diameter (approx. 47 nm) in DLS demonstrate that the above structure was actually formed (Fig. 2c), and this structure consistently exhibited an oral negative contrast effect by preserving core magnetism and suppressing aggregation and precipitation in an acidic gastric environment.

[0177] Example 3: Confirmation of core crystalline phase by XRD

[0178] The product of Example 2 was freeze-dried to obtain a powder (50 mg), and XRD (2θ) analysis was performed. Peaks at 2θ = 30.2°, 35.5°, 43.3°, 53.6°, 62.6°, and 74.2° were identified and matched JCPDS 19-0629 (magnetite, Fe3O4) (Fig. 4).

[0179] Example 4: Confirmation of Shell-Core Coordination Bond and Identification of Surface Functional Groups by FT-IR

[0180] OH (≈3225 cm⁻¹) in FT-IR (Fourier-transform infrared spectroscopy) of freeze-dried powder -1 ), CH(≒2875 / 2909 cm -1 ), C=O(asym) of COO-(≒1622 cm -1 ), CO / COC(1151 / 1104 / 1008 cm -1 ), CN(≈1558 cm -1 ) was confirmed. In addition, the COO-Fe coordination signal (≈1339 cm⁻¹) -1) and Fe-O signal (≈ 540 cm⁻¹) -1 ) was observed, proving that multi-point coordination bonds were formed between the carboxylate / hydroxyl functional groups of the shell and the iron on the core surface (Fig. 5).

[0181] Example 5: Hydration diameter and ζ-potential of the composite

[0182] INV-003 was diluted with distilled water (based on Fe 1 mg / mL) and measured by dynamic light scattering (DLS). Hydration diameter 45.0±5.0 nm (Fig. 6), ζ-potential -16.7±2.1 mV (10 mM NaCl). This is consistent with the target range for oral formulation design (total hydration diameter 10–100 nm, ζ -20–0 mV).

[0183] Example 6: Colloidal Stability (Aggregation Inhibition) - pH / Ionic Strength Durability

[0184] The colloidal stability of the iron oxide nanoparticle core-shell composite (INV-003) synthesized in Example 2 was confirmed. INV-003 (Fe 1 mg / mL) was dispersed in solutions at pH 5, 7, and 9, and in NaCl solutions at 0, 250, 500, and 1,000 mM, respectively, and the hydration diameter was observed for 7 days using DLS. A hydration diameter of approximately 45 ± 6 nm was maintained for 7 days at various pH and salt concentrations (Figs. 7b and 7c). This indicates that the iron oxide nanoparticle core-shell composite is stable without aggregation under conditions of pH 5 to 9 and NaCl 1,000 mM or less.

[0185] Example 7: Analysis of Magnetization of Iron Oxide Nanoparticles Coated with Nanostructures

[0186] The magnetization of the iron oxide nanoparticle core-shell composite (INV-003) synthesized in Example 2 was determined using a vibrating sample magnetometer (VSM). 1 mL of INV-003 was placed in a vial and dried using a freeze-dryer. 20 mg of the dried sample was placed in a sample holder, and the magnetization was measured. As shown in Fig. 8, the magnetization of the iron oxide nanoparticles (INV-003) coated with polysaccharide cross-linked colloid particles was 141 emu / g at 3 Tesla (T). Fe It was confirmed that it has the value of.

[0187] Example 8: Confirmation of T2 MRI efficacy of nanostructure-coated iron oxide nanoparticles (INV-003)

[0188] To confirm the T2MRI contrast effect of the iron oxide nanoparticle core-shell composite (INV-003) synthesized in Example 2, the T2MRI efficacy was verified using a 3 T MRI scanner. T2MRI images were taken by creating phantoms of INV-003 at concentrations of 0, 0.06, 0.03, and 0.015 mM (Fig. 9a), and the T2 relaxivity coefficient was measured by determining the 1 / T2 value for each concentration (Fig. 9b). The calculated T2 relaxivity coefficient was 217.5 mM-1s-1, confirming that it exhibits excellent T2MRI efficacy.

[0189] Example 9: Confirmation of pH-dependent stability of nanostructure-coated iron oxide nanoparticles

[0190] The stability of the iron oxide nanoparticle core-shell composite (INV-003) synthesized in Example 2 was confirmed by examining its hydration diameter and T2 MRI efficacy at different pH values. 0.05 mg of the test substance was placed in solutions at pH 0.89, 1.16, 1.48, and 1.82 (corresponding to 80, 40, 20, and 10 mM HCl, respectively), and the hydration diameter was measured using DLS for 120 minutes; the results confirmed that the substance maintained a hydration diameter of approximately 45 nm (Fig. 10a). Additionally, when the T2 relaxation time of the same sample was measured using a 0.47 T MRI scanner, it was confirmed that the substance maintained a duration of 5 ms (Fig. 10b). Furthermore, when 0.05 mg of the test substance was used as samples at pH 1.2, 3, 5, and 7 and T2 images were taken using a 3 T MRI scanner for 2 hours, no change was observed (Fig. 10c). Through this, it was confirmed that the hydration diameter and T2 MRI efficacy remain unchanged and are maintained due to changes in pH. This indicates that the contrast effect can be maintained even inside the stomach (pH 1.2) under strongly acidic conditions.

[0191]

[0192] Example 10: Mouse toxicity test of INV-003

[0193] The iron oxide nanoparticle core-shell composite (INV-003) prepared in Example 2 was administered orally and intravenously to mice, and clinical symptoms were observed. For oral administration, three mice were each administered doses of 0, 62.5, 125, and 250 mg Fe / kg; after checking for mortality, clinical symptoms (Fig. 11a), changes in body weight (Fig. 11b), and necropsy, no toxic symptoms were observed. For intravenous administration, three mice each were each administered doses of 0, 15, 30, and 60 mg Fe / kg; after checking for mortality, clinical symptoms (Fig. 12a), changes in body weight (Fig. 12b), and necropsy, no toxic symptoms were observed. Therefore, under these test conditions, the approximate lethal dose was determined to be 250 mg Fe / kg for oral administration and 60 mg Fe / kg or higher for intravenous administration.

[0194]

[0195] Example 11-1: Efficacy test of INV-003 in animal magnetic resonance cholangiopancreatography 1

[0196] The iron oxide nanoparticle core-shell composite (INV-003) prepared in Example 2 was orally administered to mice, and the nulling effect of T2 MRI signals by water in the stomach was observed. After administering the test substance at a dose of 0.67 mg Fe / kg, T2 MRI images were taken for 1 hour using a 9.4T MRI scanner (Fig. 13). As a result, a bright T2 MRI signal caused by water was observed in the stomach before administration, but it was confirmed that the T2 MRI signal was nulled (darkened) by the test substance after administration. This effect lasted for 60 minutes.

[0197] Furthermore, from the fact that the signal from INV-003 was uniform throughout the stomach, it was confirmed that even when INV-003 is magnetized by a magnetic field during MRI imaging, the particles do not aggregate due to the dextran cross-linked colloid particles in the stomach. In addition, from the fact that the signal from INV-003 persisted for 60 minutes in the stomach, it was found that iron ions were not released from INV-003 surface-modified by dextran cross-linked colloid particles in the strongly acidic stomach. Furthermore, it was found that the dextran cross-linked colloid particles are not hydrolyzed by carbohydrases in the stomach.

[0198]

[0199] Example 11-2: Efficacy test of INV-003 in animal magnetic resonance cholangiopancreatography 2

[0200] The iron oxide nanoparticle core-shell composite (INV-003) prepared in Example 2 was orally administered to pigs (body weight 40 kg) to confirm its efficacy in magnetic resonance cholangiopancreatography. After administering the test substance (INV-003) at a dose of 0.25 mg Fe / kg, T2 MRI images were taken using a 3 T MRI scanner (Fig. 14). As a result, it was confirmed that while a bright T2 MRI signal was observed in the stomach due to water before administration, the T2 MRI signal was nulled (darkened) by the test substance (INV-003) after administration. In addition, the bile ducts were observed to be brighter and clearer after the administration of INV-003, and microstructures that were not observed before administration were also observed (Fig. 14 b). In the case of the pancreatic duct, it was confirmed that some structures that were not observed due to the T2 MRI signal generated in the stomach before administration were clearly observed after the administration of the contrast agent.

[0201] Surprisingly, oral administration of 5 mL to 200 mL of a solution containing INV-003 stimulated the secretion of digestive fluid from the gallbladder, thereby enhancing the signal of the bile ducts brighter than before administration in T2-weighted images due to the digestive fluid.

[0202]

[0203] Example 12: 2-Week Repeated Oral Dose Range Finding (DRF) Test in Beagles

[0204] This example was performed to explore initial toxicity findings upon repeated oral administration of the oral administration complex (INV-003) of the present invention and to determine an appropriate dose range for the design of a subsequent 4-week repeated oral toxicity (GLP) study. Administration was performed a total of two times, on Day 1 and Day 15.

[0205] The test design was established based on the Notification of the Korea Food and Drug Administration (KFDA) on the Toxicity Test Standards for Pharmaceuticals (2022-18) and the ICH M3(R2) Guideline (2009).

[0206] Test substance: INV-003, Manufacturing number INV003S-2505, Quantitative dark brown aqueous solution of 8.0 mg Fe / mL, Store refrigerated (2-8℃).

[0207] Control: Water for injection, store at room temperature.

[0208] The formulations were prepared once on the day before administration. The target concentration formulations of 3.6 and 5.4 mg Fe / mL were prepared by diluting the stock solution of the test substance (8.0 mg Fe / mL), while the highest dose group used the stock solution without dilution. The homogeneity and stability of the formulations were confirmed for 4 hours at room temperature and 2 days under refrigeration. Concentration verification using the UV-Vis quantification method satisfied all acceptance criteria, including a recovery rate of 100±10% and a precision (RSD) within 10% (e.g., 3.6 mg Fe / mL formulation recovery rate 107.46-108.41%, RSD 0.50%; 5.4 mg Fe / mL formulation recovery rate 109.29-109.76%, RSD 0.23%). The formulations were stored under refrigeration in a light-blocked environment.

[0209] Four groups (a total of 8) of Beagle dogs, each consisting of one male and one female, were used. The administration volume was fixed at 5 mL / kg, and the reference iron (Fe) dose was set as follows.

[0210] Group 1: Control group (water for injection), 0 mg Fe / kg.

[0211] Group 2: INV-003, 18 mg Fe / kg.

[0212] Group 3: INV-003, 27 mg Fe / kg.

[0213] Group 4: INV-003, 40 mg Fe / kg.

[0214] The route of administration was oral, and it was administered once each on Day 1 and Day 15.

[0215] Unexpected mortality, general clinical symptoms, body weight and weight gain, feed intake, hematology, blood biochemistry, gross findings at necropsy, organ weight, and organ weight ratio were evaluated. All evaluations were recorded separately by sex.

[0216] No unexpected mortality was observed in any group during the observation period. No changes in clinical symptoms associated with the test substance were identified, and no dose-dependent differences were observed in body weight changes or feed intake compared to the control group. Variations in hematological and blood biochemical parameters fell within the physiological range within the species; as there were no dose-dependent or consistent changes compared to pre-administration levels, they were determined to be unrelated to the test substance. No changes related to the test substance were observed in gross findings or the organ weight-to-organ weight ratio during necropsy. Intermittently observed vomiting or changes in stool appearance (loose stool, diarrhea) were not dose-dependent and were observed as transient and sporadic, leading to the conclusion that they were unrelated to the test substance.

[0217] Under the conditions of this example, when INV-003 was administered orally twice (total of 2 times), no findings of test substance-related toxicity were observed in either male or female dogs. Therefore, it was determined that applying an iron standard of 40 mg / kg as the high dose for the 4-week repeated oral toxicity (GLP) test in Beagle dogs is scientifically valid.

[0218] This embodiment demonstrates that the orally administered complex of the present invention does not induce systemic toxicity signals in large animals (beagles) even with repeated oral exposure. This is consistent with the mechanism of action, in which the complex remains in the gastric cavity and acts locally as a T2-negative contrast agent, while the acid-resistant barrier layer and colloid stabilization of the polysaccharide-crosslinked colloid shell result in a low potential for systemic absorption and organ accumulation. These results provide valid basic data for the design of subsequent long-term (4-week) repeated toxicity studies and for the estimation of the clinical safety margin.

[0219] Example 13: Verification of the efficacy of INV-003 as an oral negative contrast agent for Magnetic Resonance Cholangiopancreatography (MRCP) in a large animal model

[0220] The purpose of this embodiment is to demonstrate, using quantitative and qualitative indicators, whether the novel contrast agent candidate INV-003 stably reduces gastric and duodenal fluid signals and improves bile and pancreatic duct contrast after oral administration to healthy miniature pigs, and to derive the optimal concentration and dosage combination per body weight for clinical application. Additionally, the effects of imaging conditions (posture, fat suppression, and degree of mixing) on ​​image quality were investigated.

[0221] 12-week-old miniature pigs weighing approximately 40 kg were used. After fasting for at least 6 hours prior to imaging, images were acquired using a respiratory-triggered 3D MRCP sequence on a clinical 3.0 T instrument under isoflurane inhalation anesthesia. In a pilot comparison, the prone position showed poor reproducibility due to increased respiratory instability and artifacts, while the supine position was established as the standard position as it was superior in terms of image quality and animal safety. Fat suppression was not applied as it was anticipated that failure of respiratory correction would lead to increased scan time and quality degradation. Oral formulations were prepared as aqueous suspensions at concentrations of 0.05, 0.10, 0.20, and 0.40 mg Fe / mL, and doses per body weight were set at 0.063, 0.125, and 0.250 mg Fe / kg for stepwise exploration. In each animal, repeated imaging was performed at 10, 30, and 60 minutes immediately after administration following non-contrast imaging, and additional delayed images were acquired at 24 hours, 7 days, and 14 days to explore excretion and accumulation. To compensate for reduced gastrointestinal motility due to fasting and anesthesia, the animals were gently shaken from side to side 6 times immediately after administration to aid in mixing the contents, and it was confirmed that the degree of gastric lumen knurling was clearly improved with 6 mixings compared to 3 mixings in the same animals.

[0222] Quantitative evaluation is the signal-to-noise ratio (SNR=SI) at the ROI set in the upper cavity. stomach / SD background ) and the noise ratio compared to each other (CNR=SI stomach -SI liver / SD backgroundChanges before and after administration were compared by calculating the values. In the concentration-dose exploration, the 0.40 mg Fe / mL formulation consistently showed the strongest negative contrast effect. When 0.25 mg Fe / kg was administered compared to 0.40 mg Fe / mL, the average SNR decreased by more than 25 to 50 times, from approximately 280–380 to 4–11, and the average CNR approached 0, decreasing from approximately 270–370 to around -1.8. At the same concentration, when 0.125 mg Fe / kg was administered, the average SNR decreased from approximately 300 to 2.1, and the average CNR decreased from approximately 234 to 0.39, confirming clinically sufficient knurling. At 0.063 mg Fe / kg, the average SNR also decreased from approximately 153 to 4.0, and the average CNR decreased from approximately 147 to -1.6. In the 0.20 mg Fe / mL formulation, SNR and CNR decreased from approximately 242 to 16 and from approximately 245 to 8, respectively, upon administration of 0.25 mg Fe / kg; the 0.10 and 0.05 mg Fe / mL formulations were excluded from clinical optimization candidates due to relatively high variability, although knurling occurred at the same dose. Reproducibility was evaluated by repeating the study with n=5 for each of the three doses (0.063, 0.125, and 0.25 mg Fe / kg) at 0.40 mg Fe / mL, and SNR and CNR significantly decreased after administration compared to before administration in all groups (pre- and post-comparison by group p<0.01). In particular, the average SNR of the 0.25 mg Fe / kg group decreased from 282.7 to 4.66 and the average CNR decreased from 276.2 to -1.76, while the 0.125 mg Fe / kg group decreased from 188.0 to 2.34 and the CNR decreased from 181.4 to -3.77, so a sufficient negative contrast effect was stably reproduced in repeated measurements.

[0223] Qualitative evaluation was conducted based on two criteria. First, when the visibility of the entire bile duct pathway was evaluated on a 4-point scale, the combination of 0.40 mg Fe / mL to 0.125–0.25 mg Fe / kg achieved "Good" or better in most cases, and "Excellent" in many instances. Second, the degree of interference with reading due to gastrointestinal high signal improved to "none" or "mild" after administration; in particular, occlusion was significantly reduced in the distal common bile duct and the section adjacent to the duodenal papilla, resulting in a distinct improvement in distal visibility. Spatial homogeneity of gastric signal suppression was maintained even in repeated imaging within the same animal, and no findings of local overconcentration due to magnetization-induced aggregation or precipitation were observed.

[0224] In repeated measures statistical analysis, no significant differences were observed between 0 hours and 7 days (p=0.581), between 0 hours and 13 days (p=0.832), and between 7 days and 13 days (p=0.489). The absence of a significant decrease in T2* over time supports the conclusion that no increase in self-sensitivity due to iron accumulation occurred, suggesting that oral administration of INV-003 is centered on local gastrointestinal action and has a low risk of accumulation in systemic organs (especially the liver). These results were consistent with the findings of no change in clinical symptoms, body weight, hematology and biochemistry, autopsy, and organ weight confirmed in previous toxicity studies.

[0225] No clinical abnormalities were observed during safety observations, and no significant changes in liver T2 or T2 were seen in delayed imaging.

[0226] The potential for excretion and hepatic accumulation was evaluated using multi-echo GRE-based liver T2* mapping. Three circular ROIs were repeatedly placed at specific locations within the liver parenchyma, and T2* values ​​were measured immediately after administration (0 hours), at 7 days, and at 13 days. The mean ± standard deviation at each time point was 4.6 ± 0.4 ms (0 hours), 4.4 ± 0.4 ms (7 days), and 4.3 ± 0.5 ms (13 days) (refer to the T2* map and bar graph in Fig. 20). Fig. 20 shows the results of quantitatively verifying the systemic absorption and organ accumulation of INV-003 through the T2* map of the ROIs set in the liver parenchyma and the change in mean T2* relaxation time. In T2* maps obtained immediately after administration (0 h), and at 7 and 13 days, there were minimal changes in the color distribution within the liver, and the mean ± standard deviation of the bar graphs and statistical test values ​​(p=0.581, 0.489, 0.832) all showed no significant difference. This indicates that the self-sensitivity (sensitivity to iron accumulation) of liver tissue did not change over time following administration. Therefore, this supports the fact that INV-003 remains and is excreted stably in the stomach and small intestine without significant absorption into the circulatory system or iron accumulation within the liver, and is consistent with safety evidence that changes in blood / hepatic iron concentrations are minimal.

[0227] Overall, oral administration of INV-003 in the miniature pig MRCP model stably reduced gastric and duodenal fluid signals by tens of times and significantly improved bile and pancreatic duct contrast, effectively resolving the gastrointestinal high-signal overlap problem, a limitation of conventional MRCP. Based on concentration-dose-reproducibility and T2 mapping results, the recommended conditions for clinical transition are to use the 0.40 mg Fe / mL formulation and select a dose within the range of 0.125–0.25 mg Fe / kg based on patient condition. It is advisable to include simple positioning (6 times left and right) immediately after administration in standard operating procedures, as this enhances the spatial homogeneity of knurling and further improves image quality. The large animal efficacy verification and liver T2* stability evaluation of this invention are consistent with the mechanism of action of the polysaccharide cross-linked colloid shell of the present invention, which simultaneously performs the functions of a strongly bound anchor ligand, acid-resistant barrier layer, microenvironment modulation, colloid and suspension stabilization in a gastric acid environment, thereby preserving core magnetism and inhibiting aggregation and precipitation, further strengthening the feasibility of developing INV-003 as an oral negative contrast agent for clinical MRCP.

Claims

1. A core composed of metal or metal oxide-based nanoparticle(s); and An oral administration complex having a hydration average particle size of 10 nm to 100 nm, comprising a shell coated with polysaccharide cross-linked colloidal particles having a hydration average particle size of 2 nm to 8 nm and a surface charge of -20 mV to 0 mV, which are disposed as an acid-resistant barrier layer on the surface of the core and form multi-point coordination bonds with metal ions on the core surface. A polysaccharide crosslinked colloidal particle for oral administration characterized in that, in an aqueous solvent, (i) the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, is modified by a first crosslinking agent having an epoxide group, (a) the functional group modified by the first crosslinking agent is directly and / or (b) two spatially adjacent functional groups modified by the first crosslinking agent are crosslinked intramolecularly and / or intermolecularly through a second crosslinking agent having two or more amine groups (-NH2) to form a polysaccharide crosslinked particle, and (ii) the surface charge of the polysaccharide crosslinked colloidal particle is within the range of -20 mV to 0 mV through modification of the number of crosslinking agent-derived basic amine groups exposed on the surface to -COOH functional groups.

2. An oral administration complex according to claim 1, characterized in that it is a mononuclear core-shell structure or a multinuclear (bridged) core-shell structure that behaves as a single hydrated particle in a dynamic fluid environment, wherein some of the polysaccharide cross-linked colloidal particles coordinately bond to one or more cores to cross-link (share) between adjacent cores, so that the number of cores of the final oral administration complex is one or more, preferably four or more, and the hydrated average particle size is 10 nm to 100 nm.

3. An orally administered complex according to claim 1, wherein the polysaccharide crosslinked colloidal particles having a hydration average particle size of 2 nm to 8 nm, which coat the core surface through coordination bonding with metal ions of metal or metal oxide-based nanoparticles, are characterized by simultaneously acting as chelators of metal ions through (i) hydroxyl groups and / or (ii) amine functional groups and / or -COOH functional groups derived from the crosslinking agent exposed on the surface where metal ions are not coordinately bonded.

4. An orally administered complex according to claim 1, characterized in that dispersion stability is maintained without aggregation or precipitation during a gastric retention of 30 to 120 minutes.

5. An oral administration complex according to claim 1, characterized in that when the solution containing the oral administration complex is administered orally, it stimulates the secretion of digestive fluid from the gallbladder.

6. An oral administration complex according to claim 1, characterized in that the surface charge of the oral administration complex is -20 mV to 0 mV due to polysaccharide cross-linked colloid particles having a surface charge of -20 mV to 0 mV coated on the core surface.

7. An oral administration structure according to claim 1, characterized in that the polysaccharide cross-linked colloid particles coated on the core surface of the oral administration complex are not hydrolyzed by carbohydrases.

8. An orally administered complex according to any one of claims 1 to 7, characterized in that 60% or more, 70% or more, 90% or more, or 95% or more of the total number of monosaccharides, which are the building blocks of polysaccharides, are modified by a crosslinking agent so as not to be hydrolyzed by enzymes in the body and so as to reduce or minimize immunostimulatory activity against immune cells.

9. An orally administered complex according to any one of claims 1 to 7, characterized in that the polysaccharide cross-linked colloidal particles are not hydrolyzed by enzymes in the body, wherein at least one -OH functional group in at least one of two consecutive monosaccharides within the polysaccharide chain is mostly modified by a cross-linking agent.

10. A core composed of metal or metal oxide-based nanoparticle(s); and A nanocomposite having a hydration average particle size of 10 nm to 100 nm comprising a shell coated with polysaccharide cross-linked colloidal particles having a hydration average particle size of 2 nm to 8 nm and a surface charge of -20 mV to 0 mV, which form multi-point coordination bonds with metal ions on the core surface, Polysaccharide crosslinked colloidal particles are formed in an aqueous solvent by (i) modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, with a first crosslinking agent having an epoxide group, (a) directly between the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group, and / or (b) intramolecularly and / or intermolecularly crosslinking between two spatially adjacent functional groups modified by the first crosslinking agent through a second crosslinking agent having two or more amine groups (-NH2), thereby forming polysaccharide crosslinked particles, and (ii) modifying the number of crosslinking agent-derived basic amine groups exposed on the surface to -COOH functional groups so that the surface charge of the polysaccharide crosslinked colloidal particles is within the range of -20 mV to 0 mV. Acid-resistant nanocomposites designed so that polysaccharide cross-linked colloidal particles simultaneously perform the functions of strongly bonded anchor ligands, acid-resistant barrier layers, aggregation inhibition, and precipitation inhibition.

11. An acid-resistant nanocomposite according to claim 10, characterized in that it is a mononuclear core-shell structure or a multinuclear (bridged) core-shell structure that behaves as a single hydrated particle in a dynamic fluid environment, wherein some of the polysaccharide cross-linked colloidal particles coordinate with two cores simultaneously to cross-link (share) between adjacent cores, so that the number of cores of the final nanocomposite is one or more, preferably four or more, and the hydrated average particle size is 10 nm to 100 nm.

12. An MRI contrast agent pharmaceutical composition comprising the acid-resistant nanocomposite of claim 10 or 11.

13. A composition for diagnosing biliary and pancreatic duct imaging containing an oral administration complex of any one of claims 1 to 9 as a contrast agent.

14. A composition for biliary and pancreatic duct imaging diagnostics according to claim 13, characterized in that the orally administered complex acts as a T2 contrast agent when it remains in the stomach during T2-weighted imaging.

15. A composition for diagnosing biliary and pancreatic duct imaging according to claim 13, characterized by confirming cancer, inflammation, gallstones, pancreatic stones, bile duct stenosis, or pancreatic duct stenosis through biliary and pancreatic duct imaging during T2-weighted imaging.

16. A composition for diagnosing biliary and pancreatic duct imaging according to claim 13, characterized in that the orally administered complex acts as a T2 contrast agent when it remains in the stomach and / or duodenum, thereby further increasing the signal of the biliary and pancreatic duct relative to the signal of the stomach during T2-weighted imaging to identify cancer, inflammation, gallstones, pancreatic stones, and biliary and pancreatic duct stenosis, and to diagnose the cause of biliary and pancreatic duct disease.

17. A method for imaging the bile duct or pancreatic duct in high contrast by orally administering an oral administration complex of any one of claims 1 to 9 to a subject, and performing T₂-weighted MRI or MRCP while the oral administration complex remains in the stomach or duodenum to reduce gastrointestinal signals.

18. Step of preparing a metal or metal oxide nanoparticle core suspension; A step of preparing a polysaccharide cross-linked colloid particle colloid solution by modifying the -OH groups of a polysaccharide in an aqueous solvent with a first cross-linking agent having an epoxide group and forming intramolecular / intermolecular cross-linking particles with a second cross-linking agent of a divalent or higher polyamine, and then partially modifying the amine groups on the surface of the polysaccharide cross-linked colloid particles with -COOH to adjust the zeta potential to -20 mV to 0 mV; and A step of mixing the above metal or metal oxide nanoparticle core suspension and the above polysaccharide cross-linked colloidal particle solution having a zeta potential of -20 mV to 0 mV. A method for manufacturing an acid-resistant nanocomposite of claim 10 or 11, comprising 19. Use in the preparation of an orally administered stabilizing modifier that induces the formation of an acid-resistant barrier, inhibition of inter-particle aggregation, and inhibition of precipitation in the gastrointestinal tract under pH 1 to 2 conditions, wherein (i) the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, is modified in an aqueous solvent by (i) modifying the -OH functional group of a monosaccharide, which is a building block of a linear polysaccharide, branched polysaccharide, or cyclic polysaccharide, with a first crosslinking agent having an epoxide group, and (a) directly between the functional group modified by the first crosslinking agent and a spatially adjacent -OH functional group, and / or intramolecularly and / or intermolecularly crosslinking between two spatially adjacent functional groups modified by the first crosslinking agent through a second crosslinking agent having two or more amine groups (-NH2), and (ii) the polysaccharide crosslinked colloidal particles, with the surface charge controlled to the range of -20 mV to 0 mV by modifying the number of crosslinking agent-derived basic amine groups exposed on the surface to -COOH functional groups.

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