Lymphangiographic contrast agent without venous contamination

Abstract

The present invention relates to a dosage form for non-vascular injection, which comprises nanoparticles whose hydrated diameter is adjusted to 2 to 20 nm and whose surface charge is adjusted to -20 mV to 0 mV by hydration of hydrophilic groups exposed on the surface so that the nanoparticles do not penetrate or are not excreted into capillaries at the injection site and are selectively excreted only into terminal lymphatic vessels, and the nanoparticles are (i) themselves a drug carrier and / or (ii) another drug carrier, and the nanoparticles are not administered intravenously but are excreted only into lymphatic vessels when injected into a tissue site, thereby providing a drug modality that extends the residence time at the injection site. According to the present invention, nanoparticles with adjusted sum diameter and surface charge can be excreted from the body without remaining at the administration site within one week after administration. When designed as a contrast agent that exhibits T1-MRI contrast effect, they can be injected into perivascular tissue sites to selectively visualize peripheral to deep lymphatic vessels without venous contamination.

Description

[Technical Field]

[0001] The present invention relates to a dosage form containing nanoparticles whose hydrated diameter is adjusted to 2 to 20 nm and whose surface charge is adjusted to -20 mV to 0 mV by hydration of hydrophilic functional groups exposed on the surface so that the nanoparticles do not penetrate or are not excreted into capillaries at the injection site and are selectively excreted only into peripheral lymphatic vessels, wherein the nanoparticles are (i) a drug itself and / or (ii) a carrier of another drug, and the nanoparticles are excreted only into peripheral lymphatic vessels when injected into a tissue site rather than intravenously, thereby providing a drug modality that extends the residence time at the injection site.

[0002] According to the present invention, nanoparticles with controlled hydrated diameters can be excreted from the body without remaining at the administration site within one week, preferably within 72 hours, after administration. When designed as a contrast agent exhibiting T1-MRI contrast effects, they can selectively visualize peripheral to deep central lymphatic vessels without venous contamination when injected into perivascular tissues. [Background technology]

[0003] Nanomaterials exhibit new physical and chemical properties that differ from bulk materials due to their reduced size. Furthermore, extensive research into nanomaterials has made it possible to control not only the size but also the composition and shape of materials, thereby realizing excellent physical and chemical properties in the nano region.

[0004] Among various nanoparticles, magnetic nanoparticles are used in a wide range of nano-biotechnologies, such as separation of biological materials, magnetic resonance imaging diagnosis, magnetic and biosensors including giant magnetoresistive sensors, microfluidic systems, drug and gene delivery, and magnetic hyperthermia therapy.

[0005] In particular, magnetic nanoparticles can be used as diagnostic agents in magnetic resonance imaging.

[0006] Magnetic resonance lymphangiography (MRL) is a medical imaging technique used to visualize the lymphatic system and its function.

[0007] Magnetic resonance lymphography (MRL) has emerged as a useful imaging technique for lymphatic imaging, taking advantage of the advantages of magnetic resonance imaging (MRI). MRL offers various advantages, including high-resolution images, 3D reconstruction, and wide body coverage, and has proven its value in post-cancer treatment, lymphatic-related surgical planning, and lymphedema monitoring.

[0008] Various contrast agents are used in MRL to further enhance contrast. Gadolinium (Gd) chelate-based contrast agents are the most widely used in clinical practice. However, their small molecular size leads to nonselective diffusion into veins, resulting in problems related to venous contamination. Because lymphatic vessels and veins cannot be visualized simultaneously, it is difficult to accurately diagnose lymphatic disorders using common Gd chelate-based contrast agents.

[0009] To address these issues, various types of nano- and macromolecular Gd-based contrast agents have been proposed, in addition to small-molecule Gd chelate-based contrast agents. However, Gd use must be limited in patients with kidney disease to prevent the risk of non-steroidal anti-inflammatory (NSF). Caution is also required because Gd can deposit in the brain and other tissues. Ultrasmall iron oxide nanoparticles, such as ferumoxytol, have demonstrated the potential for vascular imaging and could be a good alternative to Gd-based formulations. However, despite this potential, successful demonstration of ferumoxytol at standard MRLs has been rare. The only study using both Gd-based formulations and ferumoxytol showed that the Gd-based formulation enhanced both lymphatic and venous signals, while ferumoxytol neutralized venous signals. Furthermore, ferumoxytol is not FDA-approved for imaging indications due to the risk of serious hypersensitivity reactions.

[0010] Lymphatic vessels return excess interstitial fluid to the blood. Lymphatic vessels originate in the interstitium as small, thin-walled channels formed by endothelial cells that join together to form larger vessels. Initial lymphatic vessels (terminal lymphatic vessels) resemble capillaries but have many interendothelial junctions that act like one-way microvalves, also known as primary lymphatic plates. Fixing filaments connect initial lymphatic vessels to the surrounding connective tissue. The walls of larger collecting lymphatic vessels resemble those of small veins and are composed of endothelium and rare smooth muscle. Larger lymphatic vessels, like veins, have secondary lymphatic plates that limit the retrograde movement of lymph. Lymph nodes are located along the pathways of collecting lymphatic vessels. Larger lymphatic vessels drain into the left and right subclavian veins.

[0011] Lymphedema occurs when fluid and proteins accumulate in tissues due to a discrepancy between the amount of interstitial fluid passing through blood vessels and the ability of lymphatic vessels to transport this fluid. It mainly occurs in the limbs, but can occur in any part of the body, including the face, back, abdomen, chest, and genitals.

[0012] Lymphedema is the result of interstitial accumulation of lymph fluid due to damage to lymphatic vessels or lymph nodes. Imaging examinations of lymphedema are performed when lymphedema is unclear or when a more definitive diagnosis is needed to consider prognosis and treatment. However, lymphatic vessels are very small, measuring 0.4-0.8 mm in diameter, and are transparent, making them difficult to see with the naked eye. To address this issue, magnetic resonance lymphography (MRL), which uses a contrast agent to visualize lymphatic vessels and lymphedema, has become widely used clinically.

[0013] Recently, surgical treatment of lymphedema has increased, and as a result, magnetic resonance imaging (MRI) has become increasingly popular for lymphatic vessel evaluation. This field has seen many advances, from traditional T2-weighted imaging to three-dimensional imaging. Three-dimensional imaging includes spoiled gradient echo imaging, and its modified techniques are used. The mDixon technique, an essential fat suppression technique for imaging, has recently been gaining attention.

[0014] Lymphedema has been reported to occur in more than 20% of patients with breast, cervical, and ovarian cancer, and in 15.5% of all cancer patients.

[0015] Computed tomography and magnetic resonance imaging can determine the thickness of the skin, differentiate it from lipedema, identify the characteristic honeycomb shape of lymphedema, determine whether lymphatic vessels are blocked due to cancer metastasis, the degree of edema, and the degree of vascular narrowing or blockage.

[0016] In 2000, Koshima et al. published a microsurgery technique for anastomosing lymph nodes less than 0.8 mm in size with veins, and since then, surgical treatment of lymphedema has once again come into the spotlight. Recently, reconstructive surgery to alleviate lymphedema has been increasing, with lymphatico-venular anastomosis (LVA) and vascularized lymph node transfer being the most common procedures. Rapid treatment of secondary lymphedema is known to improve surgical effectiveness.

[0017] Microsurgical lymphatic reconstruction is a method of connecting lymphatic vessels and lymph nodes in areas where lymphatic stagnation occurs to normally functioning lymphatic vessels and veins. Long-term research has shown that this method is effective for limb lymphedema, but it is not widely used because it is only effective in the early stages of the disease and requires advanced microsurgical techniques.

[0018] Imaging of lymphedema is performed when lymphedema is unclear or when a more definitive diagnosis is needed to consider prognosis and treatment. Unknown causes of lymphedema include morbid obesity, dyslipidemia, endocrine dysfunction, venous insufficiency, non-recognized trauma, repeated infections, tumors, and venous malformations. The increasing number of surgeries for lymphedema is currently driving a growing demand for MRI.

[0019] Clinically available imaging techniques include bioelectric impedance spectroscopy, lymphoscintigraphy, indocyanine green lymphography (ICGL), and MRI. Direct lymphangiography was primarily used in the 1970s and 1980s, before the development of CT and MRI, to visualize lymph nodes and lymph node metastases. Lymph nodes are passageways through which bodily fluids other than blood travel. Blue dye is injected into the skin between the toes, lymphatic vessels are located, the skin is incised, and a 30-gauge needle is inserted. Iodinated oil is then slowly injected, and continuous X-rays are obtained. Images of the lymph nodes are obtained 24 hours later. However, due to the inconvenience to patients caused by invasive procedures and rare but dangerous complications such as thrombus embolism and thrombus edema, it has been replaced by modern imaging methods.

[0020] Bioelectric impedance spectroscopy measures the amount of intracellular fluid in the limbs using electrical resistance. Lymphoscintigraphy is the most commonly used diagnostic method for lymphedema and is used to observe lymphatic vessels and dermal backflow, but its diagnostic sensitivity (resolution) and specificity are lower than those of ICGL or MRI. MRI also has higher sensitivity (resolution) than lymphoscintigraphy for detecting lymphatic vessel abnormalities, while lymphoscintigraphy has higher specificity than MRI for detecting lymph nodes, revealing more lymph nodes. ICG is often used for sentinel lymph node biopsy. ICGL involves injecting ICG into the subcutaneous tissue to observe superficial lymphatic vessels with fluorescence. It is less expensive and less invasive than lymphoscintigraphy. However, MRI is more suitable than ICGL for observing lymphatic vessels located deep within the body. ICG is a contrast agent that can be directly confirmed visually in the operating room, and is therefore widely used during LVA surgery as it is suitable for real-time fluorescent imaging.

[0021] MRI is the imaging device best suited to visualizing lymphatic vessels and lymph nodes with high contrast and resolution. Lymphoscintigraphy is not suitable for viewing individual lymphatic vessels due to its low resolution, while ICGL has a penetration power of less than 2cm and is limited to observing lymph nodes near the skin. MR lymphangiography (MRL) shows lymphatic vessels regardless of depth or the state of the subcutaneous tissue. Patients with no visible lymphatic vessels often have chronic lymphedema, and in these cases, fibrosis and fatty tissue hypertrophy are visualized. MRL can be obtained with a 1.5 Tesla or 3.0T MRI.

[0022] Lymphatic vessels appear tortuous, beaded, irregular, and discontinuous in the MRL and usually appear thicker over time, distinguishing them from veins. Dermal backflow can be diagnosed as areas showing gradual, widespread diffusion of gadolinium (Gd) contrast material that penetrates the dermal tissue.

[0023] Gd-contrast agents are injected intradermally between the fingers and toes, evenly distributed across four interdigital spaces. Typically, 4–5 mL of contrast agent is mixed with 0.5–1 mL of lidocaine or mepivacaine. Gd-contrast agents are typically 0.5 mmol / mL, but higher concentrations of 1 mmol / mL have also been used. Experimental contrast agents are being studied, including gadofosveset, which, when combined with human serum albumin, has demonstrated superior performance in animal studies compared to gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) in imaging lymph nodes and lymph nodes. The gadofosveset-human serum albumin (HSA) complex is a polymeric contrast agent and is worthy of comparison with sulfur colloid and albumin used in nuclear medicine imaging. In addition, Gd-labeled dendrimers have shown good results as macromolecular contrast agents in animal imaging when used for MRL.

[0024] Recently, an imaging technique was demonstrated that uses two contrast agents, ferumoxytol and Gd-DTPA, to suppress contrast enhancement of venous blood vessels and expose only lymph nodes. Ferumoxytol is a drug developed to treat iron deficiency anemia, but due to serious hypersensitivity reactions, it requires a long injection time of over 15 minutes, which means it is not yet approved for use in imaging.

[0025] Contrast agents are drugs used to enhance the visibility of organs or tissues during imaging studies such as computed tomography (CT) scans, magnetic resonance imaging (MRI), and angiography. Contrast agents are generally approved for use in specific medical conditions or with specific imaging modalities.

[0026] Off-label use of contrast agents refers to the use of contrast agents in a manner that does not correspond to their approved indications. This can include using a contrast agent for a medical condition other than that for which it was approved, using it with a different imaging modality, or using it in a different dosage than that for which it was approved, such as by diluting it differently in a clinical setting such as a hospital. Off-label use of contrast agents is not uncommon and is useful in certain situations, such as when no other approved contrast agent is available to diagnose a particular condition. However, because the safety and efficacy of contrast agents in such situations have not been fully established, off-label use carries certain risks, such as contamination of the injectable solution by diluents and the resulting septic risk.

[0027] Currently, the following Gd-based angiographic contrast agents are used off-label for lymphography in clinical settings. In this case, the contrast agent must be diluted 10-fold or more from its existing concentration of 500 mM for lymphography. However, dispensing diluted agents in clinical settings carries the risk of dilution errors or infection due to contamination of the injection. Furthermore, unless the contrast agent is injected directly into the lymphatic vessels, its small size means that it penetrates both lymphatic and blood vessels, making it impossible to distinguish between veins and lymphatic vessels due to venous contamination (Figure 1).

[0028] [ka]

[0029] Furthermore, it has been confirmed that repeated administration of current MRI contrast agents over a long period of time increases the MRI contrast signal due to gadolinium deposition in brain tissue. Brain deposition increases in proportion to the dose and frequency of administration of the contrast agent, and can occur even in healthy individuals. It has also been reported that such deposition occurs not only in the brain, but also in bones, organs, and muscles. Linear gadolinium contrast agents are known to accumulate significantly in the body, and their use is subject to global restrictions. Summary of the Invention [Problem to be solved by the invention]

[0030] Conventional contrast agents are injected directly into lymphatic vessels before MR images are taken, but it is difficult to inject contrast agents directly into peripheral lymphatic vessels that are less than 0.8 mm in diameter.

[0031] To solve the problem of the lack of MRI contrast agents suitable for lymphatic vessels despite the urgent need for lymphatic vessel-specific contrast agents, the present invention provides nanoparticles whose hydrated diameter and / or surface charge are adjusted so that they can be injected into tissues surrounding blood vessels (FIG. 1) and selectively drained only into peripheral lymphatic vessels without penetrating or draining into capillaries at the injection site. [Means for solving the problem]

[0032] A first aspect of the present invention provides a dosage form for non-vascular injection, which contains nanoparticles whose hydrated diameter is adjusted to 2 to 20 nm and whose surface charge is adjusted to -20 mV to 0 mV by hydrating hydrophilic functional groups exposed on the surface so that the nanoparticles do not penetrate or are excreted into capillaries at the injection site but are selectively excreted only into peripheral lymphatic vessels, and the nanoparticles are (i) a drug themselves and / or (ii) a carrier of other drugs, and the nanoparticles are excreted only into lymphatic vessels when injected into a tissue site rather than intravenously, thereby providing a drug modality that extends the residence time at the injection site.

[0033] A second aspect of the present invention provides a non-vascularly injectable contrast agent composition, which contains nanoparticles having a hydrated diameter of 2 to 20 nm and a surface charge adjusted to -20 mV to 0 mV so that the nanoparticles do not penetrate or drain into capillaries at the injection site but are selectively drained into lymphatic vessels, thereby preventing venous contamination when injected into perivascular tissues and selectively imaging peripheral to central lymphatic vessels.

[0034] A third aspect of the present invention provides an information providing method characterized by providing information necessary for diagnosing lymphatic system diseases without venous contamination, using the non-vascularly injectable contrast agent composition of the second aspect.

[0035] In a fourth aspect of the present invention, there is provided a formulation for non-vascular injection containing polysaccharide cross-linked colloidal particles formed by intramolecularly and / or intermolecularly cross-linking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides with a cross-linking agent at the -OH functional groups of the monosaccharide building blocks, so as to provide a drug modality that is not administered intravenously but is drained only into peripheral lymphatic vessels when injected into a tissue site or body cavity, thereby extending the residence time at the injection site after injection to 60 minutes or more. The polysaccharide cross-linked colloidal particles have a hydrated diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and even more preferably 6 nm or less, and a surface charge of -20 mV to 0 mV, and are resistant to hydrolysis by endogenous enzymes.

[0036] The present invention will be described below.

[0037] As mentioned above, gadolinium-based contrast agents (GBCAs) are currently used for MRL. However, using GBCAs makes it difficult to visualize and differentiate between lymphatic vessels and normal veins. Furthermore, normal veins often obscure lymphatic vessels, impairing the diagnostic value of MRLs, a phenomenon known as venous contamination.

[0038] To overcome this, INV-001, the nano-MRI contrast agent used for MRL in Examples 1 to 3, is a non-gadolinium-based contrast agent consisting of a dextran core and an iron oxide shell. Because its hydrodynamic size is approximately 4 nm (smaller than the kidney-penetrating size limit), intradermally injected INV-001 is not absorbed intravenously and is absorbed only via the lymphatic system, after which it can be excreted via urine. Therefore, Example 1 confirmed the potential of INV-001 as an effective contrast agent for MRL without venous contamination to improve the diagnosis and monitoring of lymphatic status.

[0039] Specifically, the animals used in Example 1 were male Sprague-Dawley (SD) rats weighing 250-300 g. The contrast agent was injected intradermally / subcutaneously into the fourth vertebrae of both hind paws using a 30-gauge syringe. MRL was performed every 16 minutes using a 9.4-T animal imaging system using a 3D time-of-flight (TOF) sequence with a saturation band.

[0040] Unlike Gd-DOTA, which showed venous contamination in most animals regardless of injection dose and conditions, INV-001 did not. With Gd-DOTA, the popliteal lymph nodes and lymphatic vessels reached maximum enhancement at the injection site 16 minutes after injection and were quickly washed out. However, with INV-001, maximum enhancement was reached between 16 and 32 minutes after injection, allowing visualization of the popliteal lymph nodes and lymphatic vessels for a prolonged period. INV-001 at 0.45 μmol (15 mM, 30 μL) and 0.75 μmol (15 mM, 50 μL) visualized the popliteal lymph nodes and lymphatic vessels without venous contamination, interstitial space enhancement, or lymph node enlargement, and received high scores in qualitative image analysis.

[0041] Therefore, in MRL, INV-001, a new iron-based T1 contrast agent, can enhance popliteal lymph nodes and lymphatic vessels for a long period without venous contamination.

[0042] Furthermore, in Example 3, INV-001, a new type of contrast agent consisting of a dextran core and an iron oxide shell, demonstrated contrast-enhanced visualization of lymphatic vessels and nodes in beagle dogs through MRL without venous contamination.

[0043] Using healthy beagle dogs, we used the optimal administration concentration and dose for MRL of INV-001, a new type of contrast agent consisting of a dextran core and iron oxide shell, to selectively visualize only lymph nodes and lymphatic vessels, clearly demonstrating excellent MRL efficacy without venous contamination.

[0044] Quantitative and qualitative analysis revealed that the optimal concentration was 15 mM, with recommended doses of 0.056 and 0.112 mg Fe / kg for MRL. While the typical concentration of gadolinium-based MR contrast agents used clinically for MRL is 500-1,000 mmol / L and the recommended dose is 0.1-0.2 mL / kg, in Example 3, significant clinical benefits were obtained by visualizing only lymphatic vessels without venous contamination at low concentrations of 0.067 and 0.13 mL / kg. These results are of great significance to the academic community and will contribute to understanding the optimal dosing parameters for INV-001 for MRL in all clinical studies in beagle dogs.

[0045] Therefore, by using INV-001, which consists of different components (iron 0.84 mg / mL, dextran 28.0 mg / mL) from existing gadolinium-based MR contrast agents, it is possible to visualize only the lymphatic vessels without venous contamination after intradermal (intradermally / subcutaneously) administration.

[0046] In Example 7, it was confirmed that the dextran-crosslinked nanoparticles formed in Example 1 by intramolecularly and / or intermolecularly crosslinking one to three dextran or dextran derivatives with a crosslinker at the -OH functional groups of the glucose building blocks can be expanded to polysaccharide-crosslinked colloidal particles formed by intramolecularly and / or intermolecularly crosslinking one to three branched polysaccharides or two to 30 cyclic polysaccharides with a crosslinker at the -OH functional groups of the monosaccharide building blocks.

[0047] The present invention is based on this.

[0048] The formulation for non-vascular injection of the present invention contains nanoparticles whose hydrated diameter is adjusted to 2 to 20 nm and whose surface charge is adjusted to -20 mV to 0 mV by hydrating hydrophilic functional groups exposed on the surface so that the nanoparticles do not penetrate or are excreted into capillaries at the injection site but are selectively excreted only into the peripheral lymphatic vessels. The nanoparticles are (i) a drug itself and / or (ii) a carrier for other drugs, and the nanoparticles are excreted only into the lymphatic vessels when injected into a tissue site, rather than intravenously, thereby providing a drug modality that extends the residence time at the injection site.

[0049] According to the present invention, nanoparticles with hydrated diameters adjusted so that they do not penetrate or are not discharged into capillaries at the injection site and are selectively discharged only into peripheral lymphatic vessels act as an MR contrast agent, providing resolution that allows individual lymphatic vessels to be seen at the injection site regardless of the depth or state of the subcutaneous tissue (Figures 1 and 14).

[0050] Therefore, the contrast agent composition for non-vascular injection of the present invention is characterized by containing nanoparticles having a hydrated diameter of 2 to 20 nm and a surface charge adjusted to -20 mV to 0 mV so that they do not penetrate or drain into capillaries at the injection site and are selectively drained only into peripheral lymphatic vessels, thereby preventing venous contamination when injected into tissues surrounding blood vessels rather than intravenously, and selectively imaging peripheral to central lymphatic vessels.

[0051] The nanoparticles of the present invention are characterized by variously designed hydrophilic functional groups exposed on their surface, and their hydrated diameter and / or surface charge, so that they can be discharged into lymphatic vessels connected to the body's circulatory system without lymph node congestion and excreted from the body via venous blood vessels, thereby enabling them to be removed without accumulation in the body and / or without inflammatory reactions.

[0052] [Nanoparticle Modality] According to the present invention, the nanoparticles are themselves carriers of drugs and / or other drugs, with a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV adjusted by hydration of the hydrophilic functional groups exposed on the surface so that they are not discharged into the capillaries at the injection site and are selectively discharged only into the peripheral lymphatic vessels.

[0053] As used herein, a "drug" is any substance (excluding foods or devices) used to diagnose, cure, mitigate, treat, or prevent disease, or to affect the structure or function of the body, for example, any chemical or biological substance that affects the body and its metabolism.

[0054] In this specification, the term "drug" includes not only molecular level drugs but also functional nanoparticles for introduction into the body, as long as they are included in the definition of a drug. Contrast agents are also a type of drug.

[0055] As used herein, drug modality describes various methods of delivering drugs to the body, such as oral, topical, intravenous, intramuscular, subcutaneous, etc. The modality used can affect the rate at which the drug is absorbed and metabolized in the body, which in turn affects the drug's therapeutic efficacy. Thus, selecting the correct mode of administration is an important aspect of effective drug therapy or diagnostic imaging.

[0056] According to the present invention, nanoparticles whose hydrated diameter and surface charge are adjusted by hydrating the hydrophilic functional groups exposed on their surfaces so that they are not excreted into capillaries at the injection site and are selectively excreted only into peripheral lymphatic vessels can be excreted into lymphatic vessels when injected into a peripheral body site or can be directly injected into lymphatic vessels. Here, nanoparticles excreted into lymphatic vessels can be absorbed into the blood circulation system without being removed by lymphocytes and macrophages in lymph nodes.

[0057] Peripheral lymphatic vessels and capillaries are components of the circulatory system.

[0058] Peripheral lymphatic vessels are components of the lymphatic system that collect lymphatic fluid, waste, and debris in tissues and return them to the circulatory system. Peripheral lymphatic vessels are larger in diameter and thicker-walled than capillaries. Lymphatic vessels contain one-way valves that prevent backflow of fluid and facilitate its movement into the circulatory system.

[0059] In contrast, capillaries are the smallest blood vessels in the body, forming a network of tiny tubes that connect arteries and veins. Capillaries account for 90% of blood vessels (95,000 km) and connect all organs. They are responsible for the exchange of oxygen, nutrients, and waste products between blood and tissues. Capillaries have a diameter of 30 to 40 μm, with the narrowest being around 8 μm, which is just large enough for a single red blood cell to pass through, allowing a large surface area for material exchange. Because they consist of only one endothelial cell layer, material exchange with surrounding cells is easy.

[0060] The circulatory system is responsible for the flow of bodily fluids such as blood and lymph, supplying nutrients, oxygen, energy, etc. to each organ in the body, and transmitting carbon dioxide and waste products produced by life activities to the respiratory system and urinary system so that they can be excreted from the body.

[0061] Blood circulation is achieved by the action of the heart. Circulating blood performs functions such as transporting oxygen, supplying nutrients, removing waste products produced during metabolic processes, maintaining body temperature, and transporting hormones.

[0062] A large amount of ions, nutrients, organic waste products, dissolved gases, water, etc. pass through the capillaries and are mostly reabsorbed into the capillaries. The amount of body fluid that leaves the capillaries and the amount that returns to the capillaries are almost the same, and only a portion of it is absorbed through the lymphatic vessels. Here, it flows into the lymphatic vessels and returns to the blood circulation.

[0063] Lymphatic vessels are transparent, one-way tubes that transport fluids and substances primarily in the skin and submucosa to the vascular system. Lymphatic vessels are small tubes that transport lymph from tissues to lymph nodes and then back to blood vessels. After transporting lymph from tissues to lymph nodes, lymphatic vessels return fluid to the venous system via two collecting ducts. Lymphatic vessels are more permeable than capillaries and absorb macromolecules, including antigens and cells, more easily than capillaries. Lymph nodes, on the other hand, are distributed throughout the body along lymphatic vessels and connect lymphatic channels. Lymph nodes drain into collecting ducts, which then drain into two subclavian veins located below the collarbone. These veins combine to form the superior vena cava, a large vein that drains blood from the upper body to the heart.

[0064] Lymph is a pale yellow fluid that contains less protein than blood, more fat, and more lymphocytes and white blood cells. Lymph circulates throughout the body via lymphatic vessels, supplying nutrients to each cell and absorbing waste products. Lymphocytes are involved in immune responses and play a role in defending against bacteria, viruses, and other pathogens that have infiltrated the body.

[0065] After circulating throughout the body via arteries, blood flows out into the veins, where some of the body fluid remains between cells, forming interstitial fluid (tissues where plasma has been filtered and discharged through capillaries within tissues, and extracellular fluid filling the spaces between cells). When this interstitial fluid is discharged into the terminal lymphatic vessels, it is called lymph. Lymph enters the lymphatic vessels at a very slow rate and flows, finally re-entering the bloodstream. Lymph flows out of the capillary walls, cleaning the body's tissue cells and removing waste products from the tissues.

[0066] Lymph circulates throughout the body via lymphatic vessels, supplying nutrients to cells and transporting carbon dioxide (CO2) that has escaped from cells, as well as waste products such as damaged cells, cancer cells, and bacteria. After completing this process, lymph re-enters the bloodstream through the junction of the jugular vein and the subclavian vein. As lymph flows through the lymph nodes, lymphocytes in the lymph nodes react with and remove foreign substances and destroy other foreign substances. Therefore, when lymph nodes filter foreign substances that have entered from the outside, such as in acute inflammation, they swell.

[0067] Therefore, according to the present invention, nanoparticles designed to be absorbed into lymphatic vessels or injected directly into lymphatic vessels when injected into extremities of the body are preferably not removed by lymphocytes and macrophages in lymph nodes and can be absorbed into the blood circulation system.

[0068] The length of the human blood vessels is 120,000 km, and blood circulates around two and a half times the circumference of the Earth in one minute.

[0069] The nanoparticles according to the present invention are intended for in vivo injection and are therefore a type of drug.

[0070] Once a drug enters the body, the elimination process begins. The main routes of drug elimination are (1) hepatic metabolism, (2) biliary excretion, and (3) urinary excretion.

[0071] Elimination includes biotransformation (drug metabolism) and excretion, which is the removal of the intact drug from the body.

[0072] Blood flowing through blood vessels is filtered by the kidneys, and waste products are excreted in urine via the bladder. The size of the kidney's glomerular filter is generally known to be approximately 6-8 nm. Theoretically, substances within this size range or smaller should be able to pass through, but hard substances such as inorganic nanoparticles can only effectively pass through the glomerular filter if they are 5.5 nm or smaller. In other words, the size and hardness of a substance play an important role in its excretion through kidney filtration.

[0073] A high proportion of nanoparticles remaining in organs such as the liver and spleen indicates poor excretion capacity.

[0074] Therefore, the nanoparticles of the present invention are (a) polysaccharide-crosslinked colloidal particles formed by intramolecularly and / or intermolecularly crosslinking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides with a crosslinker at the -OH functional groups of the monosaccharide building blocks to prevent hydrolysis by endogenous enzymes, or (b) when surface-coated with the polysaccharide-crosslinked colloidal particles, they are squeezable during transport between internal structures due to hydration of the hydrophilic functional groups exposed on the surface. Furthermore, the nanoparticles of the present invention can be designed to selectively deliver to target sites by regulating vascular permeability and internal absorption when absorbed into the blood circulation system via lymphatics after injection. They can also be designed to regulate the availability and rate of renal excretion to control the duration or extent of blood circulation. Therefore, they are advantageous in serving as diagnostic and / or therapeutic agents for targeted diseases without accumulating in the body.

[0075] According to the present invention, nanoparticles are designed to be squeezable by hydration of the hydrophilic functional groups exposed on their surface, and have controlled hydrated diameters and / or surface charges so that they are not excreted into the capillaries at the injection site and are selectively excreted only into the peripheral lymphatic vessels, and preferably do not cause lymph node congestion. By designing these nanoparticles to be impermeable to capillaries, once they are excreted into the lymphatic vessels from the injection site and absorbed into the blood circulation system, they can be (1) metabolized in the liver, (2) excreted in the bile, and (3) excreted in the urine (Example 2) through the blood circulation, without leakage through the blood vessel wall or entering the interstitial fluid.

[0076] For example, according to the present invention, nanoparticles having a discontinuous shell in which divalent to trivalent iron ions are coordinated to hydrophilic groups derived from a crosslinker on the surface of a crosslinked dextran-based spherical core can be designed so that, after injecting into the body, they are completely absorbed into the blood circulation system through lymphatic drainage without accumulating in tissues, and can be collected in urine by stretch filtration (Example 4, Figures 6 to 10).

[0077] After administration, the nanoparticles of Example 4 were not accumulated in normal tissues and were 100% collected in the urine, indicating that they could be removed from the body only via (1) liver metabolism, (2) biliary excretion, and (3) urinary excretion.

[0078] Therefore, the nanoparticles of the present invention can be recovered through urinary excretion at a rate of 80% or more, preferably 90% or more, and more preferably 95% or more, of the amount administered into the body.

[0079] Therefore, the nanoparticles of the present invention can be collected in urine in an intact state without metabolic degradation during absorption, distribution, and / or excretion after injection into the body, and can be reused or recycled.In addition, information can be collected within the tissue, lymphatic system, or blood at the administration site by adsorption or the like.

[0080] [Nanoparticle injection site, residence time, and excretion] As shown in FIG. 1, which is a conceptual diagram showing the administration site of a contrast agent and its absorption route in the body, the nanoparticles of the present invention are not administered intravenously but are injected into the tissue surrounding the blood vessel.

[0081] The tissue surrounding the blood vessels into which the nanoparticles of the present invention are injected is connective tissue (Figure 1). Connective tissue constitutes the majority of the vertebrate body and contains cells (fibroblasts, leukocytes, and adipocytes) within an acellular extracellular matrix.

[0082] The tissues surrounding the blood vessels into which the nanoparticles of the present invention are injected are body cavities formed in epithelial tissue, such as joint cavities and intrathecal spaces.

[0083] According to the present invention, nanoparticles are hydrated to have a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV by hydrating the hydrophilic functional groups exposed on their surfaces so that they do not penetrate or are excreted from the capillaries at the injection site, but are selectively excreted only into the terminal lymphatic vessels. As a result, the nanoparticles are excreted only via the lymphatic vessels, thereby extending their residence time at the injection site and inducing interstitial space enhancement, anatomical space distension, and / or lymph node enlargement at the injection site (Examples 1 and 5).

[0084] According to the present invention, nanoparticles are hydrated to a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV by hydrating the hydrophilic functional groups exposed on the surface so that they are not discharged into the capillaries at the injection site and are selectively discharged only into the terminal lymphatic vessels. These nanoparticles are characterized by being absorbed into the body via the lymph without accumulating at the injection site, and the absorbed nanostructures are excreted from the body.

[0085] For example, the nanoparticles of the present invention can be designed so that when injected into an articular cavity or intrathecal space, they are absorbed into the blood circulation via the lymphatics and excreted in the urine by the kidneys.

[0086] The nanostructure INV-002 of Example 4 is a particle with a hydrated diameter of approximately 4 nm, in which iron is bound to a crosslinked dextran. The number-average molecular weight of INV-002 is approximately 32 kDa, approximately half that of serum albumin, and the hydrodynamic diameter is approximately 4 nm. This indicates that the size of INV-002 satisfies the size criteria required for venous drainage and is optimal for use in intra-articular injection.

[0087] An articulation is a joint between two or more bones, i.e., a region where bones come into contact and connect with each other, and is an important region that enables bodily movement. Between the bones of a joint is a thin layer of hyaline cartilage called articular cartilage. There is also a lubricating tissue consisting of a synovial membrane with a cartilage pocket-like structure and synovial cells that secrete a viscous fluid called lubricating fluid. The lubricating fluid contains proteins, salts, hyaluronic acid, etc., and provides nutrients to the joint surface and lubricates the joint. Leukocytes and lymphocytes are also present in the lubricating fluid.

[0088] The blood vessels reach the area where the fibrous layer of the joint cavity meets the lubricating membrane, and the articular cartilage is not directly supplied with nutrients.

[0089] Joint cavities are filled with synovial fluids, a viscous liquid that reduces friction between the articular cartilage of synovial joints during movement. Synovial fluid is known to contain a variety of molecules, including hyaluronic acid, proteins, and enzymes. Substances in synovial fluid can be removed by draining them into lymphatic and venous vessels that are connected to the body's circulatory system.

[0090] Although the exact size limitations for drainage are not clearly understood, the synovium is generally known to be permeable to relatively small substances such as albumin (molecular weight: approximately 66.5 kDa) and IgG (molecular weight: approximately 150 kDa). Therefore, for homogeneous distribution, effective MR arthrography, and contrast agent excretion, smaller contrast agents (<7 nm) are desired, and the nanoparticles of the present invention can be designed to simultaneously satisfy these conditions (Example 4).

[0091] Intrathecal injection is a method of injecting drugs into the spinal canal and subarachnoid space using a syringe. The spinal canal is a single tube formed by the continuous connection of vertebral foramina located in each vertebra. The spinal canal protects the spinal cord, bile duct, blood vessels, and some peripheral nerves. Intrathecal injection is also performed in the subarachnoid space, allowing the drug to reach the cerebrospinal fluid (CSF) between the tunica albuginea and pia mater.

[0092] The nanoparticles of the present invention can also be designed to enable homogeneous distribution within the spinal canal / subarachnoid space, effective MR cisternography or MR myelography, and excretion of the contrast agent upon intrathecal injection (Example 5, Figure 22).

[0093] [Nanoparticles that exert T1-MRI contrast effects] Magnetic resonance imaging (MRI) visualizes the difference in signal intensity obtained by utilizing the magnetic resonance phenomenon of hydrogen nuclei, which constitute the majority of living tissues, which differs from tissue to tissue.

[0094] MRI can measure T1 and T2 relaxations by measuring the relaxation of the hydrogen nuclear spins of water molecules.

[0095] The process by which spin nuclei (nuclear spins) excited by longitudinal magnetization are restored to their original state is called T1 relaxation or longitudinal relaxation. For T1 relaxation to occur, the energy of the hydrogen nuclei is transferred to the surrounding matter (lattice), so it is also called spin-lattice relaxation.

[0096] The substance with the longest T1 relaxation time is water, at about 3 seconds. Even if the human body does not have pure water, non-mucoid fluids move very quickly, so energy transfer is very slow and relaxation times are long. Fluids that contain only small amounts of electrolytes and no large molecules, such as cerebrospinal fluid and urine, have a very long T1 relaxation time of 2 to 3 seconds.

[0097] The relaxation time of a substance varies depending on the size of the molecule in which the proton resides. Small molecules like water move quickly, so they have low relaxation efficiency and long relaxation times. Fatty acids in adipose tissue have limited activity, so they have high T1 relaxation efficiency and the shortest T1 relaxation time of any human tissue. The T1 relaxation time increases as the magnetic field strength increases.

[0098] In the case of MRI, the contrast agent itself does not directly affect the signal, but rather the contrast agent injected into the human body penetrates into the tissue, changing the surrounding environment and altering the T1 and T2 relaxation of the tissue, thereby indirectly affecting the signal. In MRI, the magnetic material affects the relaxation time of the tissue.

[0099] MRI contrast agents are classified into T1 contrast agents and T2 contrast agents, which amplify T1 or T2 signals. T1 and T2 refer to the spin-lattice relaxation time or spin-spin relaxation time, respectively, after nuclear spin excitation in MRI, and each provides different contrast effects.

[0100] The mechanism of contrast enhancement of paramagnetic materials is that they shorten the T1 and T2 relaxation times of hydrogen nuclei present around the paramagnetic material in vivo, emitting more signals and generating contrast enhancement effects. This is due to the interaction between unpaired electrons of the paramagnetic material and hydrogen nuclei of surrounding water molecules.

[0101] In MRI, tissues with high hydrogen density, short longitudinal relaxation (T1), and long transverse relaxation (T2) appear as high signal intensity. T1 relaxation is the process by which hydrogen nuclei that absorb an RF pulse release the absorbed energy to the surroundings and return to equilibrium from the moment the RF (radiofrequency) is turned off. This energy release is fastest when the Larmor frequency matches the molecular vibration frequency of the surrounding tissue. Therefore, for an MR contrast agent to shorten the T1 of tissue, its molecular vibration frequency should nearly match the Larmor frequency, its magnetic moment should be large, and it should approach the hydrogen nuclei closely.

[0102] A typical paramagnetic substance used as an MR contrast agent is gadolinium (Gd3 + ) and the molecular vibrational frequency of gadolinium matches the Larmor frequency of the proton, so the T1 relaxation is shortened. + The electrons interact with the protons in a resonant manner, which speeds up the relaxation of the protons and shortens the longitudinal relaxation time, resulting in an increase in signal strength.

[0103] When a contrast agent is injected, it shortens the T1 of the tissue, increasing the signal intensity of the tissue. A contrast agent that causes such an effect is defined as a positive contrast agent.

[0104] T1 contrast agents consist of paramagnetic substances that undergo spin-lattice relaxation, and their presence usually produces a bright or positive contrast effect compared to water.

[0105] The nanoparticles of the present invention are designed and synthesized to be contrast agents that exhibit a T1-MRI contrast effect in which r2 / r1 is less than 5 and r1 is 1.5 or more.

[0106] To analyze whether the nanoparticles of the present invention have a contrast effect suitable for use as a T1-MRI contrast agent, the spin-spin relaxivity coefficient (r2) and spin-lattice relaxivity coefficient (r1) are measured, and their ratio (r2 / r1 ratio) is calculated. The r2 / r1 ratio is a measure of whether the nanoparticles of the present invention are suitable as a T1-MRI contrast agent or a T2-MRI contrast agent. Typical T1-MRI contrast agents have an r2 / r1 ratio of less than 5, and values ​​above 5 are difficult to use as T1-MRI contrast agents.

[0107] The nanoparticles of the present invention, which are squeezable and allow precise control of water swelling, overall size, and / or surface charge, can be observed by T1-MRI imaging for up to 1 hour after intradermal or subcutaneous injection into peripheral or central lymphatic vessels.

[0108] For this reason, when injected intradermally or subcutaneously, the administration volume is between 2 mL and 15 mL. When injected intradermally or subcutaneously, the administration volume is 2 mL or less per administration point, taking into account effective visualization and the maximum volume that can be administered intradermally or subcutaneously.

[0109] The concentration of the nano-sized magnetic substance is preferably 5 mM or more and 30 mM or less in order to obtain effective lymphatic imaging.

[0110] If an excessive amount of contrast agent is administered due to too high a concentration or volume, the contrast agent absorbed into the lymph can travel back into the veins via the thoracic duct, potentially causing the veins to be visualized.

[0111] The nanoparticles of the present invention are nano-sized magnetic substances that exhibit a T1-MRI contrast effect. Nano-sized magnetic substances that exhibit a T1-MRI contrast effect are paramagnetic or superparamagnetic.

[0112] Iron oxide nanoparticles are attracting attention as a new type of biocompatible T1 contrast agent because their magnetic properties change from superparamagnetic to paramagnetic when their size becomes extremely small (on the order of a few nanometers), resulting in an increased T1 contrast effect compared to the T2 contrast effect. Furthermore, iron oxide has fewer toxicity issues than other contrast agents, and over the long term, it can be decomposed into iron components, which can be reused in the body, thus solving the problems associated with gadolinium.

[0113] According to the present invention, a nano-sized magnetic material exhibiting T1-MRI contrast effects is, for example, manganese-iron oxide nanoparticles. Manganese-iron oxide nanoparticles can be synthesized at low temperatures below 100°C by coprecipitation in the presence of a polymer resin, which serves as a surface stabilizer for the manganese-iron oxide nanoparticles, to produce nanoparticles that exhibit monodispersity in terms of composition and / or size. Furthermore, manganese-iron oxide nanoparticles synthesized at low temperatures by coprecipitation in the presence of a surface stabilizer can be synthesized uniformly with an average diameter of 1 to 3 nm. Furthermore, the manganese-iron oxide nanoparticles can be synthesized by adjusting the manganese to iron composition ratio and / or by using various surface stabilizers for the manganese-iron oxide nanoparticles to control the distribution pattern of the manganese-iron oxide nanoparticles in the body and the possibility and degree of aggregation in the body environment.

[0114] In the present invention, nanoparticles that exhibit MRI contrast effects are (1) nanosized non-magnetic supports with iron, manganese, or gadolinium chemically bonded to their surfaces, or (2) nanosized iron, manganese, or gadolinium oxides coated with water-soluble molecules to exhibit T1-MRI contrast effects. Examples of water-soluble molecules include surface stabilizers such as polysaccharide cross-linked colloidal particles (e.g., dextran cross-linked nanoparticles (CDex)), citric acid, and polyacrylic acid (PAA), which will be described later.

[0115] Here, the non-magnetic support is a polysaccharide, a protein (e.g., albumin, aprotinin, and lysozyme), or an inorganic material (e.g., silica or gold (Au)) with hydrophilic groups exposed on its surface.

[0116] Non-limiting examples of polysaccharides include various polysaccharides containing hydrophilic chemical groups known in the art, such as dextran, cellulose, starch, glycogen, chitosan, stachyose, sucrose, xylan, araban, hexosan, fructan, galactan, mannan, agaropectin, alginic acid, carrageenan, hemicellulose, hypromellose, chitin, agarose, dextrin, carboxymethylcellulose, glycogen dextran, carbodextran, cyclodextran, fluran, or derivatives thereof.

[0117] The non-magnetic support can be made into particles by referring to various nanoparticle production methods known in the art.

[0118] For example, a method for producing silica nanoparticles is disclosed in WO2014 / 107055. Silica nanoparticles can be produced by forming reverse micelles using a suitable surfactant (e.g., a nonionic surfactant such as poly(oxyethylene) nonylphenyl ether), adding tetraethoxysilane, a silica precursor, and reacting at room temperature.

[0119] The organic polymer-based non-magnetic support can be produced by reacting an organic polymer with a crosslinking agent having a hydrophilic functional group to crosslink the organic polymer.

[0120] Crosslinking agents having hydrophilic functional groups include not only crosslinking agents that have hydrophilic functional groups in the crosslinking agent itself before reaction, but also crosslinking agents in which the chemical structure of the crosslinking agent changes after reaction to form hydrophilic functional groups.

[0121] Examples of crosslinkers include epoxide crosslinkers, amine crosslinkers, crosslinkers with anhydride groups, polyisocyanate crosslinkers, and combinations thereof.

[0122] Non-limiting examples of epoxide crosslinkers include 1-chloro-2,3-epoxypropane (epichlorohydrin), 1,4-cyclohexanedimethanol-diglycidyl ether, 1,4-butanediol diglycidyl ether, bisphenol-F diglycidyl ether, isocyanuric acid tris-(2,3-epoxypropyl)ester, neopentylglycol diglycidyl ether, triphenylolmethan triglycidyl ether, and bisphenol-A diglycidyl ether.

[0123] Non-limiting examples of amine crosslinkers include ethylenediamine, 1,3-propanediamine, diethylenetriamine, triethylenetetraamine, tetraethylenepentamine, aminoethylpiperazine, 4,7,10-trioxa-1,13-tridecanediamine, 2,2′-(ethylenedioxy)diethylamine, 1,3-bis-(aminomethyl)cyclohexane, 1,3-bis-(4-aminophenoxy)benzene, 4,4′-methylenebis-cyclohexylamine, and 5-amino-1,3,3-trimethylcyclohexanemethylamine.

[0124] Non-limiting examples of crosslinkers having anhydride groups include 2,2-bis-(4-phthalic anhydride-4-oxyphenyl)-propane, butanetetracarboxylic dianhydride, 4,4'-oxybis-phthalic anhydride, benzophenone-3,3',4,4'-tetracarboxylic dianhydride, and biphenyl-3,3',4,4'-tetracarboxylic dianhydride.

[0125] Non-limiting examples of polyisocyanate crosslinkers include 1,3-bis-(1-isocyanato-1-methylethyl)benzene, 1,3-bis-(isocyanatomethyl)-cyclohexane, hexamethylene diisocyanate, toluene-2,4-diisocyanate, trimethylhexamethylene diisocyanate, methylene di(phenylisocyanate), 4,4′-diisocyanatodicyclohexylmethane, and isophorone diisocyanate.

[0126] If the non-magnetic support is an inorganic material that does not have hydrophilic groups exposed on its surface, it can be surface-modified with water-soluble molecules that have hydrophilic groups.

[0127] The water-soluble molecules are surface stabilizers that bind to the nanoparticles through hydrocarbon chain regions and provide a surface charge to the nanoparticles through hydrophilic functional groups.

[0128] The surface stabilizer may have a hydrophilic functional group such as -COOH, -NH2, -SH, -CONH2, -PO3H, -OPO4H2, -SO3H, -OSO3H, -N3, or -NR3OH (R=C n H 2n+1 , 0≦n≦16), —OH, —SS—, —NO2, —CHO, —COX (X=F, Cl, Br, I), —COOCO—, —CONH—, or —CN, which allows the nanoparticles surface-modified with the surface stabilizers to form ionic, covalent, hydrogen, or metal-ligand coordination bonds in an in vivo environment.

[0129] Meanwhile, a method for producing iron oxide nanoparticles is shown in Example 6.

[0130] [Polysaccharide cross-linked colloidal particles cross-linked with branched or cyclic polysaccharides] According to the present invention, the nanoparticles are adjusted to have a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV by hydration of the hydrophilic functional groups exposed on the surface so that they are not absorbed or excreted in the capillaries at the injection site and are selectively excreted only in the peripheral lymphatic vessels. (a) Polysaccharide cross-linked colloidal particles formed by intramolecularly and / or intermolecularly cross-linking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides with a cross-linking agent at the -OH functional groups of the monosaccharide building blocks so as to be resistant to hydrolysis by endogenous enzymes, or (b) The surface is coated with the polysaccharide cross-linked colloidal particles.

[0131] Polysaccharides are a group of compounds consisting of multiple monosaccharides of the same or different structure linked together by enzyme-sensitive glycosidic bonds, and are widely found in the cell walls of animals, plants, and microorganisms.

[0132] These polysaccharides have a variety of physicochemical properties, such as neutral, positively or negatively charged, linear or branched molecular structures, and molecular weights ranging from several hundred to several thousand tons. These properties allow polysaccharides to have a significant effect on the biodistribution of carrier drug molecules in vivo.

[0133] The polysaccharide crosslinked colloidal particles of the present invention are obtained by crosslinking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides in an aqueous solvent using one or more crosslinking agents.

[0134] In this method, the -OH functional groups of monosaccharides, which are building blocks of branched or cyclic polysaccharides, are modified with a first crosslinking agent, and spatially adjacent modified functional groups are crosslinked intramolecularly and / or intermolecularly, either directly or with a second crosslinking agent, to form crosslinked polysaccharide particles. When an amine-based crosslinking agent is used as the first and / or second crosslinking agent, the amine groups derived from the crosslinking agent can be exposed on the surface.

[0135] The larger the molecular weight of the branched polysaccharide or cyclic polysaccharide used in the synthesis of the polysaccharide crosslinked colloidal particles, the larger the hydrated diameter (hydrodynamic size) of the polysaccharide crosslinked colloidal particles. Therefore, the hydrated diameter of the polysaccharide crosslinked colloidal particles of the present invention can be adjusted within the range of 2 to 20 nm.

[0136] The polysaccharide-crosslinked colloidal particles of the present invention, synthesized using various branched or cyclic polysaccharides, enter the blood vessels of the nephron via the circulatory system after administration to the body (e.g., intravenous injection or local administration), and are excreted in the urine by the renal filtration mechanism, as confirmed by MRI signals in the bladder (Example 7).

[0137] The branched or cyclic polysaccharides to be cross-linked are called homopolysaccharides or hetropolysaccharides.

[0138] Examples of branched or cyclic polysaccharides include dextran, cyclodextrin, maltodextrin, and inulin, as shown in FIG.

[0139] With respect to dextran crosslinked-based nanoparticles, the entire contents of Korean Patent Application No. 10-2021-0104405 are incorporated herein by reference.

[0140] Dextran is a polysaccharide derived from the condensation of glucose and is a complex branched glucan, a branched poly-α-d-glucoside of microbial origin, containing mainly C-1→C-6 glycosidic bonds.

[0141] The main chain of the polymer is composed of α(1→6) glycoside bonds between glucose monomers, and the branched portions are linked by α(1→3) glycoside bonds.

[0142] Dextran was discovered as a microbial product in wine, but mass production became possible after bacterial processes were developed. Dextran is currently produced from sucrose by certain lactic acid bacteria of the genus Lactobacillus.

[0143] Dextran is approved by the FDA as a biocompatible material.

[0144] As used herein, dextran also includes various derivatives thereof, such as carboxymethyl dextran (CM dextran), dextran sulfate, and diethylaminoethyl dextran (DEAE-dextran).

[0145] There is an increasing need for dextrans of various specific sizes in industrial applications. For example, dextrans ranging from 70,000 to 100,000 Da are used as plasma substitutes. 40,000 Da dextrans are used to reduce blood viscosity, inhibit hemagglutination, and improve blood flow. Smaller dextran sulfates of approximately 10,000 Da are used as iron transporters or anticoagulants.

[0146] Inulin is an energy-storing polysaccharide found in plants of the Compositae family, consisting of a linear chain of fructosyl groups linked by β-2,1 glycosidic bonds and terminating at the reducing end with an α-D-1,2 glucopyranoside ring. Inulin is classified as GRAS (Grade Affordable) and is used as an excipient in purified pharmaceuticals. Its water-soluble form is used to test glomerular filtration in the kidney. A specific semi-crystalline particulate form, known as delta inulin, possesses immunomodulatory properties that make it useful as a vaccine adjuvant. While inulin is indigestible by humans, intestinal bifidobacteria can metabolize it, providing probiotic effects as well as other benefits, including colon-targeted drug delivery. Modification of inulin for medical use typically involves periodic oxidation followed by functionalization via amine chemistry or reaction with anhydrides.

[0147] According to the present invention, polysaccharide cross-linked colloidal particles are formed by intramolecularly and / or intermolecularly cross-linking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides at the -OH functional groups of the monosaccharide building blocks with a cross-linking agent. The synthesis method and physicochemical properties (size, surface charge, shape, etc.) of the polysaccharide cross-linked colloidal particles can be controlled in various ways.

[0148] The polysaccharide cross-linked colloidal particles of the present invention can improve the performance of existing polymers that are widely used as coating materials for nanomaterials and pharmaceuticals in various ways by forming particles in an aqueous solution from 1 to 3 complex branched polysaccharides or 2 to 30 cyclic polysaccharides through intramolecular and / or intermolecular cross-linking with a cross-linking agent at the -OH functional groups of the monosaccharide building blocks.

[0149] According to the present invention, polysaccharide crosslinked colloidal particles, which are compact particles obtained by intramolecularly and / or intermolecularly crosslinking complex branched or cyclic polysaccharides in aqueous solution at the -OH functional groups of the monosaccharide building blocks, with a crosslinker, can be easily controlled to have a desired hydration size and water swelling degree, and can be designed to not only predictably adjust their mobility in body fluids but also be squeezable for mobility between various bodily structures. For example, in the polysaccharide crosslinked colloidal particles of the present invention, the crosslinker substitution ratio can be adjusted to 10% to 40% of the number of polysaccharide functional groups, and 2% to 98% of the crosslinkers can be adjusted so that one end does not participate in crosslinking and is exposed to the outside, thereby allowing for precise control of the water swelling degree and compressibility of the polysaccharide crosslinked colloidal particles.

[0150] Furthermore, the polysaccharide cross-linked colloid particles of the present invention can be precisely adjusted in overall size and surface charge of the polysaccharide cross-linked colloid particles and / or in vivo injection complex by adjusting at least one of the molecular weight of the polysaccharide, the length of the polysaccharide backbone, the type of cross-linking agent used during cross-linking, the amount and administration rate of the cross-linking agent administered during the synthesis reaction, and additional chemical functional group modifications, thereby ultimately imparting desired blood circulation time and desired biodistribution and excretion pharmacokinetics.

[0151] For renal excretion, the average molecular weight of the dextran or dextran derivative used is 10,000 Da or less, and the molecular weight of the spherical dextran crosslinked nanoparticles formed by crosslinking dextran-based molecules with a crosslinking agent is 35,000 Da or less.

[0152] The polysaccharide cross-linked colloidal particles of the present invention are nanoparticles in aqueous solution in which complex branched polysaccharides are intramolecularly and / or intermolecularly cross-linked with a cross-linking agent at the -OH functional groups of the monosaccharide building blocks. The branched polysaccharides can form dimers or trimers through intermolecular cross-linking, and polysaccharide cross-linked colloidal particles can be formed by controlling the intramolecular cross-linking of only one branched polysaccharide molecule with a cross-linking agent. The degree of compression can be controlled by controlling the degree of cross-linking and / or the molecular weight of the branched polysaccharide, and the desired shape is a compact sphere.

[0153] In particular, aqueous polymer solutions increase in viscosity as their concentration increases. The same is true for aqueous solutions containing polysaccharide-crosslinked colloidal particles. According to the Mark-Houwink Sakurada (MHS) equation, which relates the molecular weight and viscosity of a polymeric substance, the viscosity of a polymeric substance is known to be directly proportional to its molecular weight. If the viscosity of an injection solution is too high, it can pose risks such as vascular occlusion and vascular damage due to high pressure during injection. Therefore, in the case of drugs using the dextran-based nanoparticles of the present invention as a polyvalent linker system or drug carrier, dextran-based molecules with an average molecular weight of 10,000 Da or less, preferably about 5,000 Da or less, are used, and the molecular weight of the spherical dextran-based nanoparticles formed by crosslinking the dextran-based molecules is about 15,000 Da or less, thereby achieving a viscosity suitable for injection (in vivo administration).

[0154] For example, the smaller the average molecular weight of the branched polysaccharide or cyclic polysaccharide, the higher the concentration of the in vivo injection complex modified with the polysaccharide cross-linked colloidal particles of the present invention can be prepared. Therefore, by adjusting the degree of dilution, the viscosity of the dosage form can be precisely controlled, and the injection volume relative to the desired drug dose can be reduced.

[0155] For example, when dextran-based molecules with an average molecular weight of 10,000 Da or less are crosslinked with a crosslinker at the -OH functional groups of the glucose building blocks in an aqueous solution, one to three dextran-based molecules form spherical nanoparticles through intramolecular and intermolecular crosslinking. Therefore, the dextran-based nanoparticles of the present invention, in which the average molecular weight of the dextran-based molecules used is 10,000 Da or less, have a molecular weight of 35,000 Da or less, and the resulting modified in vivo injection complex can be realized as a nanostructure with a hydration diameter of 10 nm or less, preferably 5 nm or less, for renal excretion. Typically, nanostructures with a hydration diameter of 6 to 8 nm or less can be naturally excreted by the kidney.

[0156] Therefore, by designing various methods for synthesizing polysaccharide-crosslinked colloidal particles, including dextran-crosslinked nanoparticles, it is possible to control the distribution and excretion of functional nanoparticles or drugs in the body without immune rejection or toxicity, and in some cases, even when excreted in the body together with the polysaccharide-crosslinked colloidal particles of the present invention, they are stable without aggregation in plasma and are not metabolized or decomposed in the body after in vivo injection, allowing them to be collected through urine or feces and reused. For example, after in vivo injection, the polysaccharide-crosslinked colloidal particles of the present invention can be designed to be absorbed into the blood circulation system and excreted in urine by the kidneys without extravasation through vascular walls.

[0157] In the present invention, the crosslinking agent-derived functional groups exposed on the surface of the polysaccharide crosslinked colloidal particles are the terminal functional groups of the crosslinking agent themselves or modified or substituted functional groups thereof. For example, the crosslinking agent-derived functional groups exposed on the surface of the polysaccharide crosslinked colloidal particles are those in which at least a portion of the functional groups of the crosslinking agent exposed on the surface have been substituted with other functional groups.

[0158] In the present invention, the hydrophilic functional group may be derived from a functional group of a branched polysaccharide or cyclic polysaccharide that does not participate in the crosslinking reaction, a functional group at one end of a crosslinker that does not participate in the crosslinking reaction, and / or a functional group obtained by additionally modifying one end of the crosslinker that is exposed after the crosslinking reaction.

[0159] Examples of crosslinker-derived or hydrophilic functional groups exposed on the surface of polysaccharide-crosslinked colloidal particles include amine, carboxyl, hydroxyl, and / or thiol groups. Reactive functional groups such as amine, thiol, carboxyl, and hydroxyl groups not only facilitate surface modification but also facilitate chemical conjugation with various small molecule drugs or biopharmaceuticals such as ligands, antibodies or their fragments, antigenic peptides, and nucleic acids (e.g., DNA, RNA, or their fragments) that specifically bind to specific cell receptors. In some cases, these functional groups can be degraded by the acidic environment surrounding tumors (pH ≤ 7) or by hydrolytic enzymes to release active drugs. For example, carbonate or ester bonds are examples of acid-sensitive bonds that degrade in the acidic environment surrounding tumors (pH ≤ 7).

[0160] When the functional groups exposed on the surface of the polysaccharide cross-linked colloidal particles of the present invention are positively charged, such as amine groups, they may exhibit cytotoxicity, as with other cationic polymers. This can be resolved by substituting some or all of the amine groups with carboxyl groups, methyl groups, ethyl groups, etc.

[0161] Examples of hydrophilic functional groups that coordinate with metal ions (eg, iron ions) include amines, thiols, carboxyl groups (carboxylates and carboxylic acids), and hydroxyl groups.

[0162] The terminal hydroxyl, amine, and non-coordinating carboxylate functional groups of the polysaccharide cross-linked colloidal particles provide water solubility to the conjugate of the present invention. Therefore, hydration of the hydrophilic groups exposed on the surface of the polysaccharide cross-linked colloidal particles improves the dispersion stability of the conjugate in body fluids. This ensures the dispersion stability of the conjugate modified with the polysaccharide cross-linked colloidal particles, allowing the conjugate to which a large amount of drug is linked to be stably dispersed in body fluids without precipitation or aggregation, thereby enabling it to perform its intended function.

[0163] Furthermore, the present invention makes it possible to freely control the surface charge of the polysaccharide cross-linked colloidal particles and the surface charge of the modified object (e.g., functional nanoparticles or drugs) modified thereby by adjusting the type and / or degree of substitution of the substituents substituting the cross-linker-derived functional groups exposed on the surface of the polysaccharide cross-linked colloidal particles.

[0164] Furthermore, by adjusting the molecular weight, size, and surface charge of the polysaccharide cross-linked colloidal particles, it is possible to control the overall size and charge of the polysaccharide cross-linked colloidal particles or the in vivo injection complexes modified therewith in accordance with the desired blood circulation time, renal clearance, and hepatic clearance profiles.

[0165] According to the present invention, polysaccharide-crosslinked colloidal particles, which have been crosslinked using a crosslinking agent and optionally further modified at one end of the crosslinking agent after the crosslinking reaction, can be bound to molecules and / or drugs exhibiting targeting function via coordinate bonds, covalent bonds, hydrogen bonds, and / or electrostatic bonds through the functional groups exposed on the surface. In particular, the fact that the crosslinker-derived functional groups exposed on the surface of the polysaccharide-crosslinked colloidal particles can coordinate with metals allows various functional metal nanoparticles, which are not easily modified, to be modified to impart various physical properties. For example, divalent to trivalent iron ions can coordinate with carboxylic acids or carboxylate groups derived from the crosslinking agent on the surface of the polysaccharide-crosslinked colloidal particles. Therefore, the polysaccharide-crosslinked colloidal particles of the present invention can be used as diagnostic agents, such as contrast agents, by surface-modifying metal particles or metal oxide particles (e.g., iron oxide nanoparticles) through covalent bonds such as coordinate bonds, regardless of the synthesis method.

[0166] [Method of manufacturing nanoparticles for in vivo injection] According to the present invention, the nanoparticles have a hydrated diameter of 2 to 20 nm and a surface charge adjusted to -20 mV to 0 mV by hydration of the hydrophilic functional groups exposed on the surface so that they are not absorbed or excreted in the capillaries at the injection site and are selectively excreted only in the peripheral lymphatic vessels. The nanoparticles can contain polysaccharide-crosslinked colloidal particles formed in an aqueous solution by intramolecularly and / or intermolecularly crosslinking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides with a crosslinking agent at the -OH functional groups of the monosaccharide building blocks.

[0167] Meanwhile, the polysaccharide cross-linked colloidal particles of the present invention can be prepared, for example, by a manufacturing method including the following steps, and can be expanded to include branched polysaccharides or cyclic polysaccharides instead of dextran or dextran derivatives in the dextran cross-linked-based nanoparticles (CDex) (Example 7).

[0168] Meanwhile, the dextran crosslinked nanoparticles of the present invention can be prepared by a manufacturing method including the following steps, for example.

[0169] a first step of providing an aqueous solution of dextran or a dextran derivative; A second step of adding an alkaline aqueous solution and an epoxide dropwise at room temperature; a third step of adding two or more polyamines dropwise at room temperature to form dextran crosslinked nanoparticles having terminal amine groups; a fourth step of precipitating the product; a fifth step of redispersing the product in water; a sixth step of optionally recovering the dextran crosslinked nanoparticles having terminal amine groups by dialysis; a seventh step of administering an organic acid anhydride to the dextran crosslinked nanoparticles crosslinked with polyamines, thereby having terminal amine groups, to replace some or all of the terminal amine groups with carboxylic acid groups and / or carboxylate salt groups; an eighth step of optionally purifying the solution of dextran crosslinked-based nanoparticles having carboxylic acid and / or carboxylate functional groups to prepare water-dispersible dextran crosslinked-based nanoparticles; Optionally, a ninth step of administering an iron precursor (e.g., iron chloride) or an aqueous solution of iron oxide nanoparticles to the water-dispersible dextran crosslinked-based nanoparticles prepared in the previous step to prepare (i) dextran crosslinked-based nanoparticles on which a shell consisting of divalent to trivalent iron ions is formed, or (ii) a complex in which the surface of the iron oxide nanoparticles is modified with the dextran crosslinked-based nanoparticles; A tenth step optionally purifying or concentrating the nanostructures formed in the previous step by ultrafiltration.

[0170] In order to prevent unintended increases in hydration size due to swelling of dextran molecules, the present invention involves intramolecularly and / or intermolecularly crosslinking dextran monomers in aqueous solution. It has been discovered that when dextran is crosslinked with a crosslinking agent, two to three dextran molecules form a spherical core through intramolecular and intermolecular crosslinking. The spherical core is a crosslinked dextran-based nanoparticle formed by intramolecular and / or intermolecular crosslinking with a crosslinking agent at the -OH functional groups of the glucose building blocks within the dextran molecule.

[0171] The number of dextran molecules to be crosslinked can be controlled to be the same or different by adjusting the synthesis conditions and / or purification of the crosslinked dextran-based nanoparticles.

[0172] For example, in the second and third steps of the present invention, without the need for expensive catalysts, dextran or its derivative aqueous solution is added with (i) an epoxide that modifies the -OH functional group site of the glucose building block, and (ii) a crosslinking agent, and the reaction is carried out at room temperature to crosslink the -OH functional group of the glucose building block of dextran with the crosslinking agent, thereby producing dextran crosslinked-based nanoparticles.

[0173] In the second step, the type of epoxide is not limited as long as it modifies the -OH functional group site of the glucose building block so that it can react with the crosslinker, and is preferably a halo alkyl oxirane, such as epichlorohydrin.

[0174] In the third step, polyamines can be substituted with any crosslinking agent as long as they can covalently bond with the epoxide-derived functional groups that modify the -OH functional groups of the glucose building blocks, and this also falls within the scope of the present invention.

[0175] The second and third steps are economical because no separate expensive catalyst is required when crosslinking with a crosslinking agent at the -OH functional groups of the glucose building blocks.

[0176] The fourth step can be carried out by adding a large amount of an organic solvent having a dielectric constant of 15 to 50.

[0177] Thus, the dextran crosslinked-based nanoparticles of the present invention are synthesized in aqueous solution at room temperature without surfactants and without a ligand exchange step, and such water compatibility is advantageous for biological use.

[0178] In addition, the dextran crosslinked nanoparticles obtained by the above preparation method are in the form of a colloid dispersed in water, and depending on the degree of concentration, can be made isotonic without the need for a separate excipient. For example, the nanoparticles obtained by step 10 can be used as an injectable solution pharmaceutical composition that is non-pyrogenic and sterile without further processing.

[0179] In the ninth step, examples of hydrophilic functional groups that coordinate with the iron ion include hydroxy, carboxylic acid, carboxylate, and amine.

[0180] In addition, in accordance with the present invention, the entire contents of Korean Patent Application No. 10-2021-0104405 relating to dextran crosslinked nanoparticles having a shell made of divalent to trivalent iron ions are incorporated into the present invention and this specification.

[0181] According to the present invention, polysaccharide cross-linked colloidal particles having a shell made of divalent to trivalent iron ions are designed and synthesized in the ninth step to have the function of a T1-MRI contrast agent that shows a bright signal in MRI images, and therefore, after injection into a living body, the location of the conjugate modified with the polysaccharide cross-linked colloidal particles can be tracked by MRI images.

[0182] The polysaccharide-crosslinked colloidal particles according to the present invention can be designed and synthesized to maintain a relatively long contrast effect, thereby extending the scan time during MRI imaging and improving the spatial resolution of MRI, thereby enabling higher-resolution imaging of the vascular system throughout the body in vivo. Therefore, the polysaccharide-crosslinked colloidal particles according to the present invention are characterized by their ability to visualize microvessels, which are clinically very important but difficult to observe with existing contrast agents, in vivo using MRI. For example, imaging of a rat brain with a spatial resolution of 0.078 mm x 0.078 mm x 0.078 mm using dextran-crosslinked nanoparticles clearly revealed microvascular vessels approximately 0.078 mm thick. Considering that the spatial resolution of the 3-Tesla MRI equipment most commonly used in clinical settings is approximately 1 mm, the 0.078 mm resolution achieved by the dextran-crosslinked nanoparticles represents an approximately 13-fold improvement.

[0183] In short, the polysaccharide cross-linked colloidal particles of the present invention can also function as a T1 or T2-MRI contrast agent when the cross-linking agent-derived functional groups exposed on the surface are coordinated with divalent to trivalent iron ions.

[0184] Furthermore, in the ninth step, iron oxide nanoparticles designed to function as T1 or T2-MRI contrast agents are surface-modified with dextran crosslinked nanoparticles, making them squeezable and able to move freely between internal structures without agglomeration when injected into the body, allowing them to function as T1 or T2-MRI contrast agents.

[0185] Therefore, since the conjugate modified with polysaccharide cross-linked colloidal particles according to the present invention can serve as an MRI contrast agent, after injection into a living body, the location of the conjugate can be tracked using MRI images to confirm whether or not it is phagocytosed by macrophages, metabolically decomposed, circulated in the blood, delivered to the cellular parenchyma via capillaries, accumulated in tissues, excreted in urine by the kidneys, excreted in feces, absorbed into the vascular circulation system after injection into the living body, leaked by blood vessel walls, collected in urine / feces for reuse, and introduced into cells.

[0186] [Absorption, distribution, and excretion of nanoparticles in the human body] According to the present invention, nanoparticles with a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV are regulated by hydration of hydrophilic functional groups exposed on the surface so that they do not penetrate or are excreted into capillaries at the injection site but are selectively excreted only into peripheral lymphatic vessels. These nanoparticles are a type of drug whose synthesis method and physicochemical properties (size, surface charge, shape, etc.) can be controlled.

[0187] Pharmacokinetics, which describes the process by which the concentration of a drug found in the blood and various tissues changes over time (i.e., moves) from when the drug is absorbed and distributed in the body until it is metabolized and excreted, is the process by which the drug concentration changes over time. For example, the distribution and excretion patterns of nanoparticles in the human body are primarily determined by the physicochemical properties of nanoparticles, which are highly pure and can be precisely controlled.

[0188] After being excreted into the lymphatic vessels, the nanoparticles of the present invention can, along with the vascular circulation, (i) be absorbed by hepatocytes, secreted in the bile duct, and excreted in the feces, or (ii) be excreted by the vascular circulation system and then by the bladder into the urine.

[0189] In addition, the actions that occur when biological organs and tissues deal with foreign substances, such as nanoparticles, that have entered the body from outside, i.e., absorption in the organs, distribution to peripheral tissues via the bloodstream caused by the cardiac pulsation, and removal from the body by the liver and kidneys, cause the concentration of nanoparticles in the blood and each tissue to rise and then fall again.

[0190] Thus, the blood circulation and tissue distribution of nanoparticles not only differ depending on the various physicochemical properties of the nanoparticles, but also affect the blood circulation and tissue distribution when nanoparticles aggregate in the body.

[0191] According to the present invention, nanoparticles are controlled to have a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV by hydrating the hydrophilic functional groups exposed on their surface so that they do not penetrate or are excreted in the capillaries at the injection site, but are selectively excreted only in the peripheral lymphatic vessels. These nanoparticles can remain at the injection site for more than 2 hours and are preferably excreted in the urine by the kidneys.

[0192] In Example 4, magnetic resonance arthrography (MRA) was performed using NEMO-103 as an MR contrast agent. The results showed that joint swelling was accompanied by excellent T1 contrast effects at the joint boundary that persisted for 120 minutes, and that after 24 hours, the MR contrast agents were completely excreted via peripheral lymphatic vessels.

[0193] Examples of physical properties of nanoparticles for injecting into the body include particle size, surface charge, magnetic hysteresis curve profile, r2 / r1 value, etc.

[0194] Examples of chemical properties of nanoparticles for intracorporeal injection include the type and distribution of surface functional groups.

[0195] The size of the nanoparticles of the present invention is very important as it has a significant effect on intracellular absorption and circulation in the blood.

[0196] The size of the nanoparticles influences the extent and kinetics of accumulation.

[0197] The presence of surface charge influences nanoparticle opsonization, cell recognition by macrophages (MPS), and circulation in the bloodstream. The charge of nanoparticles is determined by the choice of material, composition, and surface stabilizers.

[0198] Therefore, by controlling the surface charge of the nanoparticles of the present invention to 20 mV to 0 mV, opsonization can be suppressed, preventing aggregation in the body and / or preventing / preventing phagocytosis by macrophages.

[0199] For example, positively charged nanoparticles have increased cell binding and uptake due to interactions between positively charged nanoparticles and negatively charged cell membranes.

[0200] The hydrophobicity of the final nanoparticles also influences their interaction with cells, while the addition of hydrophilic polymers such as PEG can retard the adsorption of blood proteins and opsonins.

[0201] As mentioned above, nanoparticles can regulate their biodistribution and excretion through 1) intravascular, 2) liver, and 3) bladder excretion via the kidneys, depending on their size, surface charge, and surface functionalization. For example, nanoparticles 6-8 nm or smaller can be excreted via the kidneys, while nanoparticles larger than that are primarily trapped in the liver. When nanoparticles have a surface charge, they form a protein corona with proteins in the blood, which increases their phagocytosis by macrophages, including Cooper cells. Conversely, the more neutral the surface charge of nanoparticles, the more inhibited the formation of a protein corona and the longer their intravascular circulation time.

[0202] The surface properties of the nanoparticles of the present invention can be controlled with precision by the type and / or density of the functional groups exposed on the surface.

[0203] On the other hand, the surface modification efficiency of nanoparticles can be estimated by measuring the chemical quantification of the functional groups, the surface charge, or the increase in surface hydrophilicity due to the hydration of surface-exposed hydrophilic functional groups. One method used to measure surface modification is to measure the zeta potential of aqueous solutions containing nanoparticles. This reflects the particle's electrical potential and is affected by the particle's composition and the medium in which the particles are dispersed. The main reason for measuring zeta potential is to predict colloidal stability. Interparticle interactions play an important role in colloidal stability. Using zeta potential measurements to predict stability quantifies such interactions. Zeta potential is a measure of the repulsion or attraction between particles due to their surface charge. Since most aqueous colloidal systems are stabilized by electrostatic repulsion, the greater the repulsion between particles, the less likely they are to approach each other and form aggregates. Because surface charge can prevent particle aggregation, nanoparticles with a zeta potential of (+ / -) 10 mV or greater are generally stable in aqueous solutions. However, nanoparticles with a large surface charge can become less stable in solutions with high concentrations of electrolyte salts, such as body fluids, as the nanoparticles can aggregate due to the electrolytes. It has been reported that nanoparticles with a large surface charge, such as amines with a positive charge and carboxylates with a negative charge, which form a type of zwitterion with a near-neutral zeta potential, are stable in aqueous solutions and in vivo conditions (blood).

[0204] Furthermore, when injected into the body, nanoparticles interact with proteins in the body through electrostatic or non-specific binding due to their surface charge. When proteins bind through this mechanism, they are likely to be recognized by immune cells. Therefore, nanoparticles need the ability to adjust their surface charge to a desired value, for example, between -20 mV and 0 mV.

[0205] As described above, the surface charge of the polysaccharide-crosslinked colloidal particles of the present invention, such as crosslinked dextran-based nanoparticles, can be precisely controlled by the type and / or density of the crosslinker-derived terminal functional groups exposed on the surface (Figure 3).

[0206] For example, a near-neutral zwitterion can be realized by combining a positively charged amine with a negatively charged carboxylate. The surface charge of the dextran crosslinked nanoparticles is -30mV to +10mV, preferably -20mV to 0mV, within the range where the terminal functional groups derived from the crosslinker are toxic and stable in body fluids.

[0207] Therefore, the nanoparticles of the present invention can provide the in vivo pharmacokinetic properties desired for the in vivo injection complex of the present invention by precisely adjusting the hydrated diameter and / or surface charge. In particular, the nanoparticles of the present invention can prevent lymph node congestion due to aggregation of nanoparticles by controlling the surface charge to 20 mV to 0 mV through hydration of the hydrophilic functional groups exposed on the surface.

[0208] The surface charge of nanoparticles not only affects their stability in aqueous solution, but also their interaction with proteins in the body. Generally, nanoparticles larger than 30 nm are easily recognized by the body's immune system and then removed by macrophages. Even if nanoparticles are small, if their surface is excessively positively or negatively charged, they can bind to blood components, mainly proteins, increasing their size and allowing them to be phagocytosed by macrophages and removed.

[0209] The surface charge of the nanoparticles of the present invention significantly affects the pharmacokinetics and behavior of the nanoparticles. For example, when serum proteins nonspecifically bind to nanoparticles via charge (i.e., opsonization), nanoparticle-protein complexes are formed, which promote phagocytosis by the mononuclear phagocyte system (MPS) and accumulation in organs. Even if nanoparticles are originally renally filterable, binding to proteins increases their size, making them unable to be filtered out by the kidney. To avoid such unintended organ accumulation and produce nanoparticles that are filtered out by the kidney, it is essential to impart properties to nanoparticles that can effectively prevent opsonization.

[0210] Therefore, in order to prevent non-specific adsorption of serum proteins, the surface charge of the nanoparticles of the present invention can be adjusted to -30 mV to +10 mV, preferably 20 mV to 0 mV, thereby regulating the blood circulation time and renal excretion.

[0211] The nanoparticles of the present invention can also induce or avoid opsonization by adjusting the surface charge to be positive, negative, or neutral (zwitterionic) using hydrophilic functional groups exposed on the surface.

[0212] The nanoparticles of the present invention can be designed to have a surface charge of −30 mV to +10 mV, preferably 20 mV to 0 mV, so as not to bind to plasma proteins such as albumin.

[0213] When nanoparticles are exposed to the in vivo environment (e.g., bloodstream, interstitial fluid, extracellular matrix), proteins form a "protein corona" on the particle surface. This corona effect causes nonspecific adsorption of proteins to the nanoparticle surface (opsonization), which determines the actual size, colloidal stability, surface properties, degree of cellular uptake, biodistribution, intracellular distribution, pharmacokinetics, tissue distribution, and toxicity of nanoparticles.

[0214] Therefore, the nanoparticles of the present invention can be substituted on the surface with water-soluble ligands or coated with amphiphilic polymers so that they show a bright T1 signal in MRI and are partially excreted by the kidney.

[0215] The nanoparticles of the present invention are designed so as not to be phagocytosed by macrophages, and can also be designed to have the function of a T1-MRI contrast agent that shows a bright signal in MRI images (Examples 1 to 7). After injection into a living body, the position of the nanoparticles of the present invention can be tracked using MRI images to determine whether they are phagocytosed by macrophages after injection, whether they are metabolically decomposed, whether they circulate in the blood, whether they circulate in the lymph, whether they are delivered to the cellular parenchyma via capillaries, whether they accumulate in tissues, whether they are excreted in urine by the kidneys, whether they are absorbed into the vascular circulatory system after injection into the living body, whether they leak through the vascular wall, and whether they can be collected in urine and reused.

[0216] The nanoparticles of the present invention can be designed so that, after injection into tissues other than blood vessels, such as articular cavities or spinal cavities, they are excreted via lymphatic vessels, absorbed into the vascular circulatory system, circulate in the blood without leakage through vascular walls, and are then excreted in the urine by renal filtration (Examples 1 to 7).

[0217] The nanoparticles of the present invention can be completely eliminated from the body, including blood vessels, joint cavities, and spinal cavities, without any injection site accumulation or adverse effects, and can be safely eliminated from the body via the renal elimination pathway. Therefore, the nanoparticles of the present invention can be injected into tissues or body cavities other than blood vessels (e.g., joint cavities, spinal cavities) and successfully demonstrate renally eliminated pharmacokinetics, thereby expanding the in vivo applicability of nanoparticles.

[0218] The nanoparticles of the present invention can be designed so that after draining through the lymphatic vessels at the injection site, they are not removed by lymphocytes in the lymph nodes as the lymph drains through the lymph nodes, and thus are absorbed into the blood circulation without causing acute inflammation that would cause swelling at the injection site and / or lymph nodes.

[0219] Furthermore, the nanoparticles of the present invention can be designed so that after in vivo administration, they are excreted in the urine in an intact state without metabolic degradation during absorption, distribution and / or excretion, and therefore can be reused or recycled.

[0220] [Surface-exposed hydrophilic groups and hydrated diameter of nanoparticles] The hydrated diameter and surface properties of the nanoparticles of the present invention can be precisely controlled by the type and / or density of the hydrophilic functional groups exposed on the surface.

[0221] Surprisingly, in lymphangiography, we found that the contrast agent designed according to the present invention can be absorbed only by lymphatic vessels without penetrating into capillaries, even when its hydrated diameter is 2-4 nm. In other words, by adjusting the hydrated diameter of the contrast agent to a minimum of 2 nm or larger, it is possible to overcome the inconvenience of the contrast agent diffusing into interstitial tissue and entering veins via capillaries, which results in the simultaneous visualization of lymphatic vessels and veins.

[0222] Therefore, the nanoparticles of the present invention can be adjusted to have a hydrated diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and even more preferably 6 nm or less, depending on the physicochemical properties (size, surface charge, shape, etc.) to provide a precisely controlled drug modality that allows selective excretion only into lymphatic vessels without venous contamination depending on the characteristics of the injection site when injected into tissues surrounding blood vessels.

[0223] Furthermore, the nanoparticles of the present invention can be designed to be squeezable by hydration of the hydrophilic functional groups exposed on their surface, so that they are not discharged into the capillaries at the injection site, but are selectively discharged only into the peripheral lymphatic vessels, and lymph node congestion does not occur.

[0224] Furthermore, the nanoparticles of the present invention can be designed to be excreted from the body within one week, preferably within 72 hours, and more preferably within 24 hours, without remaining at the injection site.

[0225] The nanoparticles of the present invention can also be designed to be excreted from the body by the liver or kidneys.

[0226] Non-vascular methods of administration of the dosage forms of the present invention are direct injection, such as intradermal, subcutaneous, or intralymphatic injection.

[0227] Therefore, when the nanoparticles of the present invention are injected intradermally or subcutaneously, the size of the injection needle that is usually used is 21 to 31 gauge.

[0228] For efficient imaging of lymphatic vessels, 1) nanoparticles must not be excreted into the capillaries at the injection site and must selectively penetrate only into the terminal lymphatic vessels for effective excretion, 2) nanoparticles that have migrated to lymph nodes must not be captured and accumulated by macrophages present in the lymph nodes, and 3) nanoparticles that have migrated to veins via the thoracic duct must not leak out of the veins again. Therefore, to satisfy these conditions, it is desirable for the hydrated diameter of the nanoparticles of the present invention to be adjusted to 2 to 20 nm based on their physicochemical properties (size, surface charge, shape, etc.).

[0229] Furthermore, the nanoparticles with a controlled hydrated diameter according to the present invention can improve drug tolerance without causing allergic reactions.

[0230] The hydrated diameter of a nanoparticle, or "hydrodynamic diameter," refers to the size of a particle in solution and is affected by factors such as particle shape, surface charge, and interactions with other particles and the surrounding solvent. This diameter can be measured using various techniques, such as dynamic light scattering and nanoparticle tracking analysis. The hydrated diameter is an important property of nanoparticles because it influences their behavior in biological systems, including their transport and distribution in vivo, their interactions with cells and tissues, and their elimination from the body. The hydrated diameter also influences the pharmacokinetics and pharmacodynamics of drugs encapsulated within nanoparticles. Therefore, accurately determining and controlling the hydrated diameter of nanoparticles is important for drug development and delivery.

[0231] Nanoparticles vary in size depending on the organs they are taken into and their distribution in the human body. Nanoparticles larger than 50 nm are phagocytosed by Kupffer cells in the liver and quickly accumulate in the liver.

[0232] However, even if the nanoparticles are small, if they are not dispersible, they easily solidify and can be removed in large quantities by the reticuloendothelial system (RES).

[0233] For example, nanoparticles with a hydrodynamic diameter of 1 to 30 nm can remain in the blood for a long period of time without being engulfed by phagocytes. Furthermore, nanoparticles with a size of 6 to 8 nm or less can be naturally excreted via the kidneys. Nanoparticles with a diameter exceeding this value are difficult to excrete via the kidneys from the body. Therefore, when the nanoparticles of the present invention are excreted into urine via the kidneys, they can be designed to have a total hydrated diameter of 8 nm or less, preferably 6 nm or less, and a uniform size distribution.

[0234] Conversely, suppressing excretion through urination increases the residence time in the bloodstream, allowing the blood drug concentration to be maintained at a high level for a long period of time, and increases the efficiency of delivery to the reticuloendothelial system, improving drug delivery to organs with a well-developed reticuloendothelial system, such as the liver and spleen. To increase the residence time of the nanoparticles of the present invention in the bloodstream or increase the efficiency of delivery to the reticuloendothelial system, the nanoparticles can be designed to have a uniform size distribution with a total hydrated diameter of 8 nm or more.

[0235] [Capillary impermeability] According to the present invention, the nanoparticles are squeezable due to the hydration of the hydrophilic functional groups exposed on their surface, and have a hydrated diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and even more preferably 6 nm or less, and a surface charge adjusted to -20 mV to 0 mV, so that they are not discharged into the capillaries at the injection site but are selectively discharged only into the peripheral lymphatic vessels, preventing lymph node congestion. These nanoparticles are impermeable to capillaries due to the hydration of the hydrophilic functional groups exposed on their surface.

[0236] Capillary structures vary widely in the basement membrane compartment, with slit junctions between endothelial cells. In the liver and spleen, most of the basement membrane is exposed by large, discontinuous capillaries, allowing large plasma proteins to pass through. The capillary structure of the brain is continuous and lacks slit junctions. Closely juxtaposed cells form tight junctions that constitute the blood-brain barrier (BBB). To enter the brain, drugs must pass through or be actively transported through capillary endothelial cells in the central nervous system (CNS).

[0237] The size of the gaps (pores) between cells that make up the blood vessel wall is known to be approximately 2 nm, and various substances smaller than 2 nm can be extravasated. Therefore, vascular permeability can be controlled by adjusting the size of the substance. Through size control and surface charge, the nanoparticles of the present invention can be designed to be impermeable to capillaries so that they can be excreted into the lymphatic vessels from the injection site and absorbed into the blood circulation system, and then circulate in the blood without leakage through the blood vessel wall or entering the interstitial fluid.

[0238] The chemical properties of a drug strongly influence its ability to cross cell membranes. Lipid-soluble drugs are dissolved in lipid membranes and can pass through all cell surfaces. Hydrophilic drugs, on the other hand, do not easily cross cell membranes and must pass through slit junctions.

[0239] The nanoparticles according to the present invention are formed so that one end of the cross-linker having a hydrophilic functional group exposed on the surface of the polysaccharide cross-linked colloidal particle, or a functional group derived from the cross-linker, is exposed to an aqueous environment. Therefore, the nanoparticles are hydrophilic drugs and can be designed to not only not pass through cell membranes but also slit junctions between endothelial cells in capillaries, i.e., to be capillary impermeable.

[0240] For example, the polysaccharide cross-linked colloidal particles of the present invention can be designed so that they are filtered by the kidney but do not permeate the blood vessel walls of normal capillaries and are excreted only into the lymphatic vessels.

[0241] Healthy capillaries, which are impermeable to the polysaccharide cross-linked colloid particles of the present invention, have a continuous endothelium with tight junctions and a basal lamina. Therefore, the polysaccharide cross-linked colloid particles of the present invention, which are designed to be filtered in the kidney, can be filtered in the capillaries of the liver and spleen.

[0242] The polysaccharide cross-linked colloidal particles of the present invention are filtered by the kidney, but do not pass through the vascular walls of normal capillaries and are excreted only into lymphatic vessels. The hydrated diameter of the polysaccharide cross-linked colloidal particles of the present invention is 2 to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, and the -COOH functional group is exposed on the surface, and the surface charge can be designed to be within the range of -20 mV to 0 mV.

[0243] The polysaccharide cross-linked colloidal particles of the present invention, when administered locally, can extend their residence time at the injection site, thereby inducing interstitial space enhancement, anatomical space distension, and / or lymph node enlargement at the injection site. Furthermore, the extended residence time at the target administration site can enhance the efficacy of the drug (Examples 1 to 5).

[0244] The polysaccharide cross-linked colloidal particles of the present invention, when designed to have a hydrated diameter of 10 nm or less, preferably 8 nm or less, and more preferably 6 nm or less, and a surface charge of -20 mV to 0 mV so that they can be filtered by the kidney, can be circulated through the vascular system and then excreted in urine via the kidney, resulting in an excretion time (90% or more) of 48 hours or less, preferably 24 hours or less.

[0245] The polysaccharide cross-linked colloidal particles of the present invention are designed to be easily excreted in urine, are not metabolized in the liver, and are not hepatotoxic.

[0246] For example, when the hydrated diameter of polysaccharide cross-linked colloidal particles is 2 nm to 10 nm, preferably 8 nm or less, and more preferably 6 nm or less, and the surface charge is adjusted to the range of -20 mV to 0 mV, the particles are filtered by the kidney but are excreted only into the lymphatic vessels without passing through the vascular walls of normal capillaries. Therefore, when administered locally, the particles are easily excreted in urine, which makes it possible to control side effects due to organ retention or accumulation (Examples 5 and 7).

[0247] For example, by adjusting at least one of the molecular weight of polysaccharides such as dextran or dextran derivatives (e.g., carboxymethyldextran), the length of the polysaccharide backbone, the type of crosslinking agent used in crosslinking, the amount and administration rate of the crosslinking agent administered during the synthesis reaction, and chemical functional group modification, the molecular weight and surface charge of the polysaccharide crosslinked colloidal particles, the final size and charge of the polysaccharide crosslinked colloidal particles can be adjusted, and they can be designed so that they are not phagocytosed by macrophages or are phagocytosed by macrophages, and so that they are excreted in the urine or feces via the excretory action of the kidney or liver. Furthermore, desired blood circulation time, or desired biodistribution and excretion pharmacokinetics can be imparted.

[0248] [Nanoparticles that are not excreted into capillaries and are selectively excreted only into peripheral lymphatic vessels] The nanoparticles of the present invention can be designed so that they are not excreted into the capillaries at the injection site and are selectively excreted only into the peripheral lymphatic vessels by adjusting the hydrated diameter to 2 to 20 nm and the surface charge to -20 mV to 0 mV through hydration of the hydrophilic functional groups exposed on the surface.

[0249] The lymphatic system is an essential part of the immune system and includes organs such as the thymus, bone marrow, spleen, tonsils, appendix, and Peyer's patches in the small intestine, which produce and process specialized white blood cells that fight infection and cancer. The lymphatic system consists of (i) thin-walled lymphatic vessels, (ii) lymph nodes, and (iii) two collecting ducts.

[0250] Lymphatic vessels are distributed throughout the body and are larger than capillaries (the smallest blood vessels connecting arteries and veins) but smaller than the smallest veins. Most lymphatic vessels have valves, just like veins, to allow the condensed lymph to flow in one direction (towards the heart). Lymphatic vessels drain a body fluid called lymph from tissues throughout the body, and after transporting lymph from tissues to lymph nodes, they return the fluid to the venous system via two collecting ducts.

[0251] Lymph begins as fluid that diffuses into the spaces between cells through the very thin walls of capillaries. Most of the fluid is reabsorbed by the capillaries, and the rest is drained into the lymphatic vessels and eventually returned to the veins. Lymph also contains (i) proteins, minerals, nutrients, and other substances that allow the fluid to provide nourishment to tissues, and (ii) many other substances, including damaged cells, cancer cells, and foreign bodies (e.g., bacteria and viruses) that have entered the tissue fluid.

[0252] Lymph nodes are small, bean-shaped organs that act as collection centers for lymph. All lymph travels through strategically located lymph nodes, filtering out damaged cells, cancer cells, and foreign particles. Lymph nodes also contain specialized white blood cells (e.g., lymphocytes and macrophages) that ingest and destroy damaged cells, cancer cells, infectious organisms, and foreign particles. Thus, an important function of the lymphatic system is to remove damaged cells from the body and prevent the spread of infection and cancer. Some lymph nodes are clustered under the skin, particularly in the neck, armpits, and groin. Other lymph nodes are located deep within the body, for example, in the abdomen.

[0253] Lymph nodes drain into collecting ducts, which in turn drain into the two subclavian veins located below the collarbone. These veins join to form the superior vena cava, the large vein that drains blood from the upper body into the heart.

[0254] The nanoparticles of the present invention can be designed to be injected into a peripheral region of the body and absorbed into the lymphatic vessels, or can be injected directly into the lymphatic vessels. In this case, when injected into a peripheral region of the body or directly into the lymphatic vessels, they flow into the lymphatic vessels and can be absorbed into the circulatory system without being removed by lymphocytes and macrophages in the lymph nodes.

[0255] Furthermore, when the nanoparticles of the present invention function as an MRI contrast agent, they can be absorbed / injected into lymphatic vessels and used to perform MRI imaging of the presence or absence of lymphatic vessel abnormalities in the lymphatic circulation pathway, such as lymphatic vessel blockage, lymphedema, swollen lymph nodes, lymphadenitis, lymphoma, and migration of tumors in other organs to lymph nodes near the tumor.

[0256] [Contrast agents used in medical imaging procedures to visualize the lymphatic system and its functions] The non-vascularly injectable dosage form of the present invention provides a contrast agent for medical imaging procedures used to visualize the lymphatic system and its function.

[0257] Here, the nanoparticles contained in the non-vascular injectable formulation of the present invention serve as a contrast agent for diagnosing diseases and effectively imaging biological phenomena at the molecular and cellular levels.

[0258] The nanoparticles of the present invention can also be excreted into lymphatic vessels with a diameter of 0.1 mm or more.

[0259] By using nanoparticles, whose physicochemical properties (size, surface charge, shape, etc.) can be controlled in various ways, as imaging agents, it is possible to predict various interactions in the internal physiological environment, for example, at the molecular, cellular, and organ levels, from the captured images. This makes it possible to predict the chemical or physical functions of individuals, organs, cells, and biochemical molecules present in the living body.

[0260] Furthermore, according to the present invention, the nanoparticles are squeezable and have a controlled hydrated diameter due to hydration of the hydrophilic functional groups exposed on their surface so that they are not discharged into the capillaries at the injection site but are selectively discharged only into the peripheral lymphatic vessels, thereby preventing lymph node congestion. This allows for evaluation of lymph flow, drainage, and congestion.

[0261] According to the present invention, nanoparticles are squeezable and have a controlled hydrated diameter due to hydration of the hydrophilic functional groups exposed on their surface so that they are not discharged into the capillaries at the injection site but are selectively discharged only into the peripheral lymphatic vessels, thereby preventing lymph node congestion.When used as a contrast agent, these nanoparticles can be injected into the tissue surrounding blood vessels rather than intravenously, allowing for selective visualization of peripheral to central lymphatic vessels without venous contamination.

[0262] Peripheral lymphatic vessels and central lymphatic vessels are two types of lymphatic vessels that play important roles in the lymphatic system. Peripheral lymphatic vessels are located in tissues throughout the body and serve to collect excess fluid and debris from tissues and transport them to central lymphatic vessels. These lymphatic vessels are small in diameter and have thin, semipermeable walls that allow fluid and debris to enter but not exit. Peripheral lymphatic vessels merge to form larger lymphatic vessels as they progress toward the deep lymphatic vessels.

[0263] In contrast, deep lymphatic vessels are larger lymphatic vessels located near the heart that carry lymph from the peripheral lymphatic vessels into the bloodstream. These lymphatic vessels have thick walls and high pressure, which helps pump lymph along the lymphatic network. Deep lymphatic vessels also contain specialized valves that prevent backflow of lymph and regulate the flow of lymph through the lymphatic system.

[0264] When the nanoparticles of the present invention are magnetic nanoparticles, they can be used as a diagnostic tool in magnetic resonance imaging.

[0265] MRI imaging has the advantage of excellent soft tissue contrast and no radiation-related risks.

[0266] Medical imaging techniques that use contrast agents to visualize the lymphatic system and its functions include magnetic resonance lymphangiography (MRL), ultrasound lymphangiography, computed tomography (CT) lymphangiography, positron emission tomography (PET) lymphangiography, and lymphoscintigraphy.

[0267] The choice of imaging technique will depend on a variety of factors, such as the characteristics of the condition being evaluated, the patient's general health, and the availability of equipment and expertise.

[0268] [Dosage form] "Drug dosage form" refers to the process of combining various chemical and physical components to produce a final drug or pharmaceutical product. Drug dosage form is an important aspect of drug development and determines a drug's stability, bioavailability, and efficacy. The formulation process involves selecting appropriate ingredients and determining optimal ratios to create a safe and effective final product. Components used in drug dosage forms include active ingredients, excipients, and other substances that improve the drug's stability, solubility, and bioavailability. Dosage form can affect the physical properties of a drug, such as appearance, taste, and texture, which affect patient acceptance and compliance.

[0269] Different types of dosage forms are used for different routes of administration, such as oral, topical, intravenous, intramuscular, and subcutaneous. The dosage form can affect the drug's release profile, i.e., the rate at which the drug is released into the body and the duration of effect. Therefore, drug dosage forms must be carefully considered to achieve optimal therapeutic results and minimize potential side effects.

[0270] According to the present invention, nanoparticles with a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV are hydrated by hydrating the hydrophilic functional groups exposed on the surface so that they are selectively excreted only into the peripheral lymphatic vessels and not into the capillaries at the injection site. These nanoparticles can be formulated without the need for a separate dilution formulation to achieve optimal contrast effects (Example 5). Therefore, because they are provided as a diluted vial product, they are relatively free from problems such as infection, contamination, and heterogeneous contrast agent concentrations.

[0271] According to the present invention, nanoparticles are hydrated to a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV by hydrating the hydrophilic functional groups exposed on their surface so that they are not excreted into capillaries at the injection site and are selectively excreted only into terminal lymphatic vessels. These nanoparticles can be administered by direct injection into lymphatic vessels, subcutaneous injection, intramuscular injection, body cavity injection, endothelial administration, topical administration, intradermal administration, pulmonary administration, and rectal administration.

[0272] In the present invention, the non-vascular injection method is preferably intradermal injection, subcutaneous injection, or direct intralymphatic injection.

[0273] Subcutaneous injections provide a more rapid effect than oral administration, but drug absorption is slower than intramuscular injections, with the absorbable dose being approximately 2 cc or less. Good blood circulation has the advantage of almost complete absorption into the tissues, and the duration of action is generally within 30 minutes.

[0274] On the other hand, intradermal injection is a method of injecting a small amount of injection solution into the dermis, the layer just below the epidermis. The dermis has a small blood supply and slows absorption, so it is used in case a severe allergic reaction occurs due to rapid drug absorption. Small amounts of drug, typically less than 0.1 ml, are administered. Because the dermis has no blood vessels, absorption is the slowest among parenteral administrations. Incorrect dosage can be dangerous, and inflammation, bleeding, and allergic reactions can occur at the injection site.

[0275] [MR Technique] An MRI sequence is a specific set of MRI pulse sequences and pulsed field gradients used to obtain a specific image. Gradient-echo (GRE) sequences are used for T1-weighted imaging, and fast spinecho (FSE) sequences are used for T2-weighted imaging.

[0276] When the nanoparticles of the present invention are contrast agents that exhibit T1-MRI contrast effects, the non-vascularly injectable dosage form of the present invention can be used in magnetic resonance imaging lymphangiography.

[0277] Magnetic resonance imaging lymphography uses the largest phased-array coil to acquire images. Images of the lower extremities are acquired in four sections, from the feet to the pelvis, and images of the upper extremities are acquired in three sections, from the hands to the axilla. However, depending on the capabilities of the machine and coil, images of the lower extremities are often acquired in three sections up to the groin. The latest equipment has developed coils that cover the entire lower extremity, making examinations easier, but when using only one coil, images should be acquired in sections.

[0278] The acquisition of high-resolution isotropic images is important in MRL, and post-processing involves multiplanar reformation and maximum intensity projection (MIP) for image evaluation. MIP images are particularly essential for lymphatic vessel evaluation. MRL primarily acquires isovoxel images as 3D images, occasionally acquiring 2mm slices in the coronal plane. When axial images are reconstructed using 3mm slices, they are reconstructed in the axial or coronal planes to provide anatomical information and displayed as basic images. Isovoxel has the advantage of being reconstructed in multiple planes.

[0279] Lohrmann et al. reported that the strongest contrast enhancement was observed in the lower limbs 45 minutes after contrast injection and in the upper limbs 55 minutes after. Furthermore, while veins exhibit weaker signal intensity over time, lymphatic vessels exhibit stronger signal intensity over time. According to Liu et al., the diameter of lymphatic vessels was less than 2 mm in 31%, 2-5 mm in 50%, and 5-8 mm in 19%. The number of lymphatic vessels observed was 1-2 in 40%, 3-20 in 36%, and 20 or more in 24%.

[0280] Traditional MRL images include heavy T2-weighted turbo spin echo images (hereinafter referred to as T2WI), fat-suppressed T1-weighted 3D spoiled gradient-echo images (hereinafter referred to as SPGR, GE) or fast low-angle shots (hereinafter referred to as FLASH, SIEMENS), and T1-weighted fast-field echo images (hereinafter referred to as T1FFE, Philips).Heavily T2WI is a method that has been used for a long time and is also used in 3D T2WI images to show lymphatic vessels filled with fluid.Subcutaneous injection of Gd-based agents has limited use, and although it has the advantage of being able to be seen without the injection of contrast agents, it has limitations in use because it cannot see normal lymphatic vessels and can only see dilated lymphatic vessels.

[0281] T1-weighted 3D images, due to the characteristics of contrast-enhanced MRL images, show lymphatic vessels well. However, they are not widely used because they take approximately 30 minutes to acquire an image of the entire upper or lower limb. Intermediate-weighted 3D images are more limited in showing lymphatic vessels than 3D T1WI images.

[0282] SPGR and FLASH are traditionally widely used methods and have been used in many papers. Volumetric interpolated breath-hold examination (VIBE) and Controlled Aliasing in Parallel Imaging Results in Higher Acceleration (CAIPIRINHA) are primarily used in abdominal and thoracic MRI to evaluate central lymphatic vessels. VIBE is the same technique as SPGR, and CAIPIRINHA is a method for reducing aliasing artifacts in 3D images, demonstrating improved signal-to-noise and artifact reduction compared to existing parallel techniques. VIBE is a modified version of FLASH, and it has been reported that VIBE provides superior image quality for peripheral veins compared to FLASH. In VIBE images, the short repetition time causes almost all tissues to appear dark, while contrast agents exhibit strong signal intensity.

[0283] MR lymphangiography requires fat suppression, but uniform fat suppression is necessary because non-uniform fat suppression can mask lymphatic enhancement and produce poor images. The mDixon method is the latest fat suppression imaging technique and is useful for uniform fat suppression. Images obtained by applying the mDixon method to existing T1FFE images have recently attracted attention. The acquisition times for VIBE, CAIPIRINHA, FLASH, and T1FFE-mDixon methods are similar. In the author's experience, the T1FFE-mDixon method, which produces sufficient SNR with a 1mm isovoxel and exhibits uniform fat suppression, is the most advanced method, and its use is expected to expand.

[0284] MRL is the most effective imaging method for visualizing lymphedema and lymphatic vessels, and its use has been increasing recently. T2WI is limited to viewing edema and anatomical information, and 3D images are the basic imaging method for MRL. It has evolved from SPGR to its modified methods, VIBE, CAIPIRINHA method, and T1FFE-mDixon method. MIP is an essential method for MRL, allowing you to see lymphatic vessels at a glance.

[0285] [Lymphedema and lymphatic system disorders] The nanoparticles of the present invention have a hydrated diameter of 2 to 20 nm and a surface charge of -20 mV to 0 mV adjusted by hydration of the hydrophilic functional groups exposed on their surface so that they do not penetrate or are excreted in the capillaries at the injection site and are selectively excreted only in the peripheral lymphatic vessels. When the nanoparticles contain at least one component selected from the group consisting of iron, manganese, and gadolinium, they can function as a contrast agent for disease diagnosis and effectively imaging biological phenomena at the molecular and cellular levels. Furthermore, the non-vascular injection dosage form of the present invention can be used to evaluate lymph flow, drainage, and congestion.

[0286] Thus, the non-vascularly injectable contrast agent composition of the present invention can visualize lymphatic vessels, lymph glands and / or lymph nodes.

[0287] Furthermore, when the nanoparticles of the present invention function as an MRI contrast agent, they can be absorbed / injected into lymphatic vessels and used for MRI imaging of lymphatic vessel abnormalities in the lymphatic circulation pathway, such as lymphatic vessel blockage, lymphedema, swollen lymph nodes, lymphadenitis, lymphoma, and migration of tumors in other organs to lymph nodes near the tumor.

[0288] Furthermore, the non-vascularly injectable contrast agent composition of the present invention can be used to provide information necessary for diagnosing lymphatic diseases without venous contamination. Here, angiography is eliminated before nanoparticles are discharged into veins via lymphatic vessels, thereby eliminating angiographic noise. Therefore, lymphatic vessel obstruction images can be provided. Furthermore, information necessary for the prevention or early diagnosis of lymphedema can be provided.

[0289] The non-vascularly injectable dosage form of the present invention preferably uses nanoparticles designed to prevent dermal backflow or lymph node congestion in the lymphatic vessels at the site of lymphedema.

[0290] Lymphedema occurs when the volume of interstitial fluid filtered through capillaries and entering the primary lymphatic vessels exceeds their lymphatic transport capacity. Because primary lymphatic vessels lack muscle cells other than valves that direct lymph in one direction, they act like a pump, transporting lymph to secondary lymphatic vessels, due to deformation of surrounding tissues caused by factors such as muscle contraction and relaxation in the limbs and skin compression and relaxation. Lymphatic pumps can also function through minute movements such as heartbeat, breathing, and organ movement. Lymphedema can occur when interstitial fluid levels increase due to cardiac arrhythmias, thrombosis, varicose veins, hypoalbuminemia, or heart failure, or when lymphatic transport capacity is reduced due to congenital lymphatic vascular development problems, lymphatic vessel damage caused by surgery, radiation therapy, infection, or trauma, or reduced lymphatic pump function due to prolonged floating. Prolonged lymphedema can lead to tissue fibrosis and disfigurement. Depending on the severity of the edema, the patient may feel heavy, stiff, and in pain. This reduces the patient's movement, which reduces the lymphatic pumping function and progressively worsens the edema due to the reduced lymphatic transport capacity.

[0291] Treatment becomes difficult as lymphedema progresses over time and subcutaneous fibrosis progresses, making prevention and early diagnosis important. When a patient presents with lymphedema, a medical history, physical examination, and basic screening tests (general blood tests, liver function tests, kidney function tests, electrolyte tests, albumin tests, thyroid function tests, fasting blood glucose tests, inflammation tests (erythrocyte sedimentation rates, C-reactive protein), brain natriuretic peptide, hepatitis tests, urinary tests, chest radiography, electrocardiogram, etc.) should be performed to identify systemic diseases and medications that may be causing or worsening the edema. If the cause of lymphedema is unclear, the cause should be differentiated using bioelectrical resistance tests, Doppler ultrasound, lymphangiography, computed tomography (CT), magnetic resonance imaging (MRI), genetic testing, and dermatophyte infection tests.

[0292] Peripheral lymphedema is a disease that improves with medical treatment, but it often progresses to chronic edema, causing repeated infections, changes like elephant skin, serious disabilities, and even fatal sequelae such as lymphangiosarcoma (Stewart-Treves syndrome). Peripheral lymphedema occurs primarily in association with breast cancer and gynecological cancer treatment. The incidence rate of upper limb lymphedema after breast cancer surgery ranges from 2 to 65%, but it mainly occurs after lymph node resection or lymph node radiation therapy, with the incidence rate in gynecological cancers ranging from approximately 11.1 to 50%.

[0293] Lymph node removal or radiation treatment destroys or obstructs central lymphatic vessels, inhibiting afferent drainage and increasing intralymphatic pressure, resulting in pathological changes in lymphatic vessels. This leads to lymphedema, which causes accumulation of protein-rich fluid in the interstitial tissue, inducing an inflammatory response, promoting adipocyte proliferation and fibrous tissue deposition, and increasing vulnerability to infection.

[0294] The lymphatic system not only plays a passive role in transporting fluids, fatty acids, and immune cells, but also an active role in regulating the immune response. It is also affected by changes in the immune microenvironment, and a dysfunctional immune response can induce lymphatic system dysfunction. [Effects of the Invention]

[0295] According to the present invention, nanoparticles with regulated hydrated diameters can be excreted from the body within one week after administration without remaining at the administration site. When designed as a contrast agent that exhibits T1-MRI contrast effects, they can selectively visualize peripheral to central lymphatic vessels without venous contamination when injected into tissues surrounding blood vessels.

[0296] The lymphographic contrast agent according to the present invention can highlight lymphatic vessels and evaluate lymphatic flow, drainage, and congestion. MR lymphography can provide important information in certain cases, particularly in the evaluation of lymphatic system diseases such as lymphedema and preoperative assessment of lymphatic vessel damage and lymphatic drainage patterns. In these situations, MR lymphography can be useful in guiding clinical decision-making and treatment planning. [Brief explanation of the drawings]

[0297] [Figure 1] 1 is a schematic diagram showing the lymphatic or venous penetration pathway of a contrast agent after intradermal / subcutaneous injection, and a schematic diagram showing the principle of lymphatic selective imaging of a lymphatic contrast agent without venous contamination according to the present invention. [Figure 2] Figure 1 shows the experimental workflow of Example 1. (a) Magnetic resonance lymphangiography (MRL) timeline, (b) methylene blue staining performed 2 days after MRL (yellow triangle: popliteal lymph node (LN)), (c) ROIs designated for analysis (red circle: popliteal LN, blue circle: muscle, yellow circle: background, red arrow: lymphatic vessel, blue arrow: muscle, yellow arrow: background). [Figure 3]Figures showing the characteristics of INV-001 used in Example 1. (a) The structure of INV-001 consists of a dextran core and an iron oxide shell. (b) Hydrodynamic size of INV-001. (c) T1-WI, T2-WI, T1, and T2 mapping images of an INV-001 serially diluted phantom. (d) T1 and T2 relaxation rates as a function of concentration, and linear fitting. The slope of the line indicates r1 and r2. [Figure 4] This figure compares the effects of Gd-DOTA and INV-001 on popliteal lymph nodes (LN) and lymphangiography in Example 1. (a) 3D-TOF scan images before (0 min), 16 min, and 96 min after intradermal injection of Gd-DOTA and INV-001. (b) Signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) analysis of popliteal LN and lymph vessels (LV) after administration of Gd-DOTA and INV-001. [Figure 5] Figure 1 compares the presence or absence of venous contamination during administration of Gd-DOTA and INV-001 in Example 1. Images of MRL using Gd-DOTA and INV-001 and lymphatic confirmation photographs using methylene blue staining were taken. Visualization of lymph nodes and lymphatic vessels 16 minutes after injection of various concentrations into the hind paw. (a, e) Gd-DOTA and INV-001 2.25 μmol (75 μL, 30 mM), (b, f) 1.125 μmol (75 μL, 15 mM), (c, g) 0.45 μmol (30 μL, 15 mM), (d, h) methylene blue staining. Yellow triangles, yellow arrows, and red arrows indicate lymph nodes, lymphatic vessels, and saphenous vein, respectively. [Figure 6]

[0023] Figure 1 shows the results of a dose optimization study to improve interstitial space and minimize lymph node enlargement in Example 1. Various concentrations and doses were injected into the hind paw, and popliteal LN and LV visualization was performed 16 minutes later. [Figure 7]This figure shows the qualitative scores of Gd-DOTA and INV-001 in Example 1. (a, b) Qualitative scoring results for each group under various administration conditions. (c) MRL image 16 minutes after Gd-DOTA and INV-001 injection and methylene blue staining. The yellow triangle, yellow arrow, and red arrow indicate the lymph node, lymphatic vessel, and saphenous vein, respectively. [Figure 8] This figure shows the results of comparing the concentration and viscosity at maximum concentration of the nanostructure for intradermal / subcutaneous / intravenous injection (code name INV-001) synthesized in Example 2 and the nanostructure for intra-articular injection (code name INV-002). [Figure 9] FIG. 1 shows images of the MRL of mouse lower limbs taken at 16-minute intervals for 96 minutes after administration of INV-001. [Figure 10] This figure shows confirmation of venous contamination using methylene blue (left side) and confirmation of the presence or absence of venous contamination using MRL images taken after intradermal administration of INV-001 and Dotalem (right side). [Figure 11] The results show that all of the administered INV-001 was excreted from the MRL images of the mouse lower limbs taken 24 hours after intradermal administration of INV-001. [Figure 12] FIG. 1 shows the experimental design for dose determination in the beagle dog MRL experiment using INV-001. [Figure 13] FIG. 1 shows the setting of the region of interest (ROI) for SNR and CNR analysis in an MRL experiment in beagle dogs using INV-001. [Figure 14] FIG. 1 shows MRL imaging results for dose setting in a beagle dog MRL experiment using INV-001 at doses of 0.028 mg / kg (a), 0.056 mg / kg, and 0.112 mg / kg. [Figure 15] FIG. 1 shows the results of quantitative analysis of SNRLN, SNRLV, CNRLN, and CNRLV values ​​over time according to the administered dose and volume in an MRL experiment in beagle dogs using INV-001. [Figure 16]FIG. 1 shows MRL images demonstrating reproducibility at doses of 0.056 and 0.112 mg / kg in an MRL experiment in beagle dogs using INV-001. [Figure 17] FIG. 1 shows the values ​​of SNRLN, SNRLV, CNRLN, and CNRLV over time, based on quantitative analysis results showing reproducibility at doses of 0.056 and 0.112 mg / kg in a beagle dog MRL study using INV-001. [Figure 18] 13 shows a photograph of the intradermal administration of INV-001 contrast agent to the lower limb of a rat using a 31-gauge syringe in Example 2-13. [Figure 19] FIG. 1 shows the administration site during intradermal administration in an MRL experiment using INV-001 in beagle dogs. [Figure 20] In an MRL experiment using INV-001 in beagle dogs, the T1(A), T2(B), and T2*(C) relaxations observed in the liver and kidney before and 48 hours after administration were the same before and after administration, indicating that the administered substance was completely excreted without accumulation. [Figure 21] In an MRL experiment using INV-001 in beagle dogs, MRL images taken before administration, 30 minutes after administration, and 48 hours after administration showed that 48 hours after administration, no INV-001 remained at the administration site and it had been completely excreted. [Figure 22] These are efficacy data from Radd magnetic resonance arthrography (MRA) performed after injection of INV-002 into the Radd joint cavity. (a, b) Schematic diagram of intra-articular tissue structure. (c, d) MRI images of the Radd joint in the INV-002-injected experimental group and the saline-injected control group. (e) Comparison of T1-MRI signal intensity before and after INV-002 injection, and (f) comparison of CNR of intra-articular tissue. [Figure 23]These are the efficacy data of Ladd magnetic resonance imaging arthrography performed after injection of INV-002 into the Ladd joint cavity. (a, b) Ladd joint MRI images of the experimental group injected with INV-002 and the control group injected with gadolinium contrast agent (Dotarem). (c) A graph comparing the T1-MRI signal intensity of the joint cavity over time after injection of INV-002 and gadolinium contrast agent (Dotarem). [Figure 24] (a) Comparison of iron content in urine collected after INV-002 injection into the Rudd joint cavity and urine collected after saline injection, and (b) comparison of urine sample color. [Figure 25] Trace iron analysis data in the Ladd joint cavity using Perls Prussian blue immunohistochemical staining. (a) Micrographs of Ladd joint cavity tissue injected with INV-002 and (b) of Ladd joint cavity tissue injected with saline. [Figure 26] FIG. 1 shows T1-MRI images over time after INV-002 is injected into the Radd spinal canal. [Figure 27] FIG. 1 shows data on body weight patterns measured for 14 days after injection of INV-002 into the Rudd joint cavity. [Figure 28] FIG. 1 shows blood chemistry data analyzed after injection of INV-002 into the Rudd joint space. [Figure 29] The structure of NEMO-103 (a), a dextran crosslinked-based T1-MRI contrast agent, its MRI phantom image (b), and its relaxivity (d) compared with that of Dotarem (c, e), a representative gadolinium-based contrast agent (GBCA) for clinical use in T1-MRI. [Figure 30] This figure shows the phase division by imaging time of magnetic resonance arthrography (MRA) performed for a clinical comparison study between NEMO-103 (iron-based contrast agent, IBCA), a dextran crosslinked-based T1-MRI contrast agent, and GBCA. [Figure 31]FIG. 1 shows the method for a clinical comparative study of NEMO-103, a dextran crosslinked-based T1-MRI contrast agent, and GBCA. [Figure 32] This figure shows MRA images taken 30 minutes (a) and 60 minutes (b) after administration of NEMO-103 to a human body, and a comparison of the CNR (c), Distance (d), and Overall quality (e) of these images. [Figure 33] This figure shows MRA images taken 30 minutes after administration of NEMO-103 (a) and gadolinium contrast agent (GBCA, b) to a human body, and a comparison of the contrast-to-noise ratio (CNR, c), distance (d), and overall quality (e) of these images. [Figure 34] This figure shows MRA images taken approximately 60 minutes after administration of NEMO-103 (a, c) and GBCA (b, e) to the human body, and a comparison of the CNR (c), Distance (d), and Overall quality (e) of these images with GBCA. [Figure 35] FIG. 1 shows the surface charge of C-DNPs synthesized by adjusting the amount of succinic anhydride used to replace the functional groups exposed on the C-DNP surface with carboxyl groups. [Figure 36] This figure shows an electron microscope image of iron oxide nanoparticles (IONPs) (a), the size of iron oxide nanoparticles observed by electron microscope (4 nm, b), the size of iron oxide nanoparticles coated with carboxyl group-substituted C-DNP-3, C-DNP-5, and C-DNP-10 (c), and the size of iron oxide nanoparticles coated with C-CMDNP-100 (d). [Figure 37] FIG. 1 shows the surface charge of iron oxide nanoparticles coated with C-DNPs with tuned surface charge. [Figure 38] Figure 1 shows the colloidal stability (a) and hydrated diameter change (b) of C-DNP-coated iron oxide nanoparticles over time under various physiological conditions (salt, pH). [Figure 39] FIG. 1 shows the colloidal stability of iron oxide nanoparticles depending on the type of dextran and C-DNP used for coating (a), and the hydrated diameter depending on the amount of dextran used for coating (b). [Figure 40] As an example of the present invention, the electrophoresis results showing the anti-opsonization effect of C-DNP-5 coated iron oxide nanoparticles with surface charges of +5 mV (a), -3 mV (b), and -20 mV (c) are shown. [Figure 41] MRI images showing the pharmacokinetics of C-DNP-coated iron oxide nanoparticles designed by adjusting the size and surface charge of the C-DNP to have the following properties: (a) 7 nm hydrated diameter / surface charge -3 mV, (b) 11 nm hydrated diameter / surface charge -3 mV, and (c) 11 nm hydrated diameter / surface charge -30 mV. [Figure 42] 1 shows the structural formula of an example of a branched polysaccharide or cyclic polysaccharide to be crosslinked in a polysaccharide crosslinked colloid particle. [Figure 43] This figure shows the crosslinking of Dextran T-5 (a), Maltodextrin (b), α-cyclodextrin (c), β-cyclodextrin (d), and Inulin (e) confirmed using a UV-Vis spectrophotometer. [Figure 44] This figure shows MRI phantom images of materials synthesized by introducing iron into crosslinked bodies of Dextran T-5 (a), Maltodextrin (b), α-cyclodextrin (c), β-cyclodextrin (d), and Inulin (e), as well as r1 and r2 relaxivity (f). [Figure 45] This figure shows T1-MRI images (a) taken after intravenous administration of a substance synthesized by introducing iron into crosslinked bodies of Dextran T-5, Maltodextrin, α-cyclodextrin, β-cyclodextrin, and Inulin to mice, and the signal-to-noise ratio (SNR) before and after administration (b). [Figure 46]This figure shows T1-MRI images confirming renal excretion after intravenous administration of substances synthesized by introducing iron into crosslinked bodies of Dextran T-5 (a), Maltodextrin (b), α-cyclodextrin (c), β-cyclodextrin (d), and Inulin (e) to mice. DETAILED DESCRIPTION OF THE INVENTION

[0298] The present invention will be described in more detail below with reference to examples, which are provided to clearly illustrate the technical features of the present invention and are not intended to limit the scope of the present invention.

[0299] Example 1: Magnetic Resonance Lymphography This example compares the pharmacokinetics of gadolinium-based and iron oxide-based contrast agents inside the lymphatic structure using magnetic resonance lymphangiography (Comparison of the Pharmacokinetics of Gadolinium-Based and Iron Oxide-Based Contrast Agents inside the Lymphatic Structure using Magnetic Resonance Lymphangiography).

[0300] Gadolinium (Gd)-based contrast agents are primarily used in contrast-enhanced magnetic resonance lymphography (MRL). However, overcoming the issue of venous contamination remains a challenging task. This study aimed to evaluate the MRL efficacy of a newly developed iron-based contrast agent (INV-001), specifically designed to mitigate the issue of venous contamination. We also sought to explore the optimal dose, including injection volume and concentration, required for successful visualization of popliteal lymph nodes and surrounding lymphatic vessels.

[0301] 1-1. Preparation of Gd-DOTA and INV-001 The Gd-based contrast agent used was Gd-DOTA (Gd-DOTA, DOTAREM, Guerbet, Roissy CdG Cedex, France). The Gd-DOTA stock solution, provided at a concentration of 500 mM, was diluted with saline prior to administration.

[0302] INV-001 was synthesized as follows: 20 mM dextran T5 (average molecular weight 5,000 Da) solution was mixed with sodium hydroxide solution and epichlorohydrin, followed by the addition of diethylenetriamine. The cross-linked dextran was purified using a 3-kDa cutoff ultrafiltration filter. The terminal amine was additionally modified with a carboxyl functional group. After ultrafiltration, the carboxylated dextran was reacted with iron chloride solution for 1 hour, and the final product was purified and concentrated by ultrafiltration.

[0303] 1-2. Hydrodynamic size and MR relaxivity of INV-001 The hydrodynamic size of INV-001 was analyzed and measured to be 3.6 nm (Fig. 3b). The T1 and T2 relaxation rates (1 / T1 and 1 / T2) of INV-001 were then measured at 9.4 T (Fig. 3c). The T1 and T2 relaxation coefficients (r1 and r2) were calculated at various INV-001 concentrations (0.125, 0.25, 0.5, and 1.0 mM). r1 was 2.61 mM. -1 s -1 , r2 is 4.33mM -1 s -1 (Fig. 3d, e).

[0304] The INV-001 contrast agent is larger than 2 nm, the maximum size for intravasation, and cannot penetrate into veins. Therefore, in principle, it can be used as a lymphatic vessel-specific contrast agent that penetrates into lymphatic vessels without venous contamination when injected intradermally or subcutaneously.

[0305] 1-3. Comparison of SNR and CNR between INV-001 and Gd-DOTA The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) of the popliteal lymph node and surrounding lymphatic vessels were analyzed with 1.125 μmol INV-001 (75 μL, 15 mM) and 1.125 μmol Gd-DOTA (75 μL, 15 mM). Sequential MRI imaging revealed the dynamic patterns and peak time points of SNR and CNR of the popliteal lymph node and lymphatic vessels (Figure 4a). In the Gd-DOTA 1.125 μmol group, peak enhancement of the popliteal lymph node was observed at 16 minutes (SNRLN, 26.96 ± 10.78; SNRLV, 11.15 ± 5.83; CNRLN, 21.61 ± 10.04; CNRLV, 7.89 ± 5.15 with 1.125 μmol Gd-DOTA). The INV-001 1.125 μmol group showed peak enhancement between 16 and 32 min during prolonged visualization (SNRLN, 23.49 ± 8.64; SNRLV, 16.05 ± 3.52; CNRLN, 15.87 ± 11.05; CNRLV, 12.58 ± 3.37) with 1.125 μmol INV-001; Figure 4b).

[0306] 1-4. Observation of venous contamination, increased interstitial space, and enlarged lymph nodes After injection of contrast agent into the hind paw, MRL was performed using 3D TOF under saturation band conditions to distinguish lymphatic vessels. For Gd-DOTA, venous contamination was observed in the 2.25 μmol (75 μL, 30 mM) and 1.125 μmol (75 μL, 15 mM) groups, except for the 0.45 μmol (30 μL, 15 mM) group (red arrows in Figures 5a and 5b). Popliteal lymph nodes and lymphatic vessels were contrasted in all INV-001 groups without venous contamination (yellow arrows in Figures 5e, 5f, and 5g). Methylene blue was injected 2 days after MRL to confirm the corresponding structures (Figures 5d and 5h).

[0307] 1-5. Qualitative image analysis of various injection parameters Various conditions and qualitative images were analyzed using Gd-DOTA and INV-001. In all groups, MRL demonstrated favorable visualization of the popliteal lymph nodes and lymphatic vessels (Figure 6). Qualitative image analysis was performed according to Table 1 (Criteria for qualitative score).

[0308] [Table 1]

[0309] INV-001 0.75 μmol (75 μL, 10 mM), 1.125 μmol (75 μL / 15 mM), 1.5 μmol (75 μL, 20 mM), and Gd-DOTA 2.25 μmol (75 μL, 30 mM) administered at adjusted injection concentrations received scores of 4.2, 5, 5, and 3.8, respectively (Figure 7a). INV-001 0.3 μmol (20 μL, 15 mM), 0.45 μmol (30 μL, 15 mM), 0.75 μmol (50 μL, 15 mM), and Gd-DOTA 2.25 μmol (75 μL, 15 mM) administered at adjusted injection concentrations received scores of 3.6, 4.4, 4.6, and 3.2, respectively (Figure 7b). Overall, the qualitative image analysis results for INV-001 showed that lymphatic vessels and lymph nodes were clearly visible without venous contamination or skin reflux, with 0.45 μmol (15 mM, 30 μL) and 0.75 μmol (15 mM, 50 μL) achieving the highest scores of 4.4 and 4.6, respectively (Figure 7b and Figure 7c).

[0310] [Discussion] INV-001 is an iron-based nano-MRI contrast agent specifically designed for MRL. In phantom studies, INV-001 has demonstrated T1 contrast effects similar to Gd-based contrast agents (INV-001 ≈ 2.6 mM). -1 s -1 vs. Gd-DOTA ≒ 3.7 mM -1 s -1) Subsequently, an in vivo comparison of INV-001 and a Gd-based contrast agent (Gd-DOTA) showed that administration of 1.125 μmol of INV-001 demonstrated relatively high CNR and SNR in the popliteal lymph nodes and surrounding lymphatic vessels. INV-001 was shown to enhance the popliteal lymph nodes for a longer period than Gd-DOTA.

[0311] Such delayed washout of INV-001 suggests that the time window for scanning may be wider than that of Gd-DOTA.

[0312] Unlike Gd-DOTA, which showed intravenous penetration at doses above 0.45 μmol in most animals, INV-001 did not show intravenous penetration, suggesting that this unique property of INV-001 stems from its optimal hydrodynamic size (3.6 nm), which limits intravenous penetration.

[0313] The size of a contrast agent has been reported to determine its pharmacodynamic and pharmacokinetic properties, and small molecules are rapidly cleared at the MRL. Gd-based contrast agents, with a size of 1 nm (molecular weight <1,000 g / mol), can be rapidly washed out of the veins, causing venous contamination.

[0314] Finally, 24 and 48 hours after injection, no INV-001 was found to remain at the injection site, lymphatic vessels, or lymph nodes. Furthermore, statistical analysis of the T1, T2, and T2* relaxation times of the liver and kidney compared with pre-injection values ​​revealed no significant differences. This suggests that INV-001 was excreted from the body within 24 and 48 hours (data not shown).

[0315] In conclusion, this example demonstrated the potential of INV-001 as a T1 contrast agent for MRL.

[0316] Therefore, INV-001 can potentially be used as a T1 imaging agent to improve the diagnosis and monitoring of lymphatic diseases.

[0317] [Conclusion] This example demonstrates that INV-001, a novel contrast agent, provides long-term improved visualization of popliteal lymph nodes and lymphatic vessels without venous contamination. By optimizing injection parameters, INV-001 effectively visualized popliteal lymph nodes and lymphatic vessels, with the highest scores achieved at 0.45 μmol (15 mM, 30 μL) and 0.75 μmol (15 mM, 50 μL). These results demonstrate the potential of INV-001 for MRL.

[0318] Example 2. 2-1. Preparation of contrast agent for MR lymphangiography 180 μmol of dextran (average molecular weight 5,000 Da) was dissolved in 9 mL of distilled water, and epichlorohydrin and NaOH were added. Then, 380 mmol of diethylenetriamine was added, and the mixture was stirred at room temperature (RT) for 24 hours. After 24 hours of succinylation at room temperature, the dextran core was purified by dialysis using a 5 kD molecular weight cutoff (MWCO) filter. An excess of iron chloride hexahydrate solution was added to the dextran core solution. The pH was adjusted to 8 using 1 M NaOH, and the mixture was reacted at room temperature for 1 hour. After purification by dialysis, the nanostructure (code name: INV-001) was synthesized.

[0319] The maximum concentration of INV-001 was 2.6 mg / mL, with a viscosity of 5.3 cP (Figure 8).

[0320] The hydrated diameter of the synthesized INV-001 is 3.9 nm (surface charge is -3.3 mV). The r2 / r1 ratio measured by 3T MRI is 1.22 (r1 = 3.57 mV). -1 s -1 , r2=4.35mM -1 s -1), which is very close to the theoretical ideal T1-MRI contrast agent value of 1, making it suitable as a T1-MRI contrast agent. This contrast agent is larger than 2 nm, the maximum size for intravasation, and is therefore unable to penetrate into veins. Therefore, in principle, it can be used as a lymphatic vessel-specific contrast agent that penetrates into the lymphatic vessels without venous contamination when injected intradermally or subcutaneously.

[0321] The contrast agent is prepared by dispensing it at concentrations of 10 mM, 15 mM, and 20 mM based on the metal standard, and an appropriate amount of mannitol is added to make it an isotonic solution, adjusting the osmolality to 280-340 mOsm / kg.

[0322] 2-2. Contrast injection method for MR lymphangiography INV-001 was administered intradermally between the toes of the animals' lower paws using a 31-gauge syringe in a volume of 10, 20, 30, 40, 50, or 75 microliters per animal, which corresponds to 2, 4, 6, 8, 10, or 15 mL in human doses.

[0323] 2-3. Animal preparation for MR lymphangiography Before MRI, the animals are anesthetized with isoflorane (0.5-2% concentration, delivered with oxygen). If necessary, local anesthesia with lidocaine can be used to reduce pain.

[0324] 2-4. MR Lymphangiography Imaging Method Before the injection of the contrast agent, MRI was performed to obtain a baseline image, and after the injection of INV-001 in Example 2-1, imaging was performed at 16-minute intervals for 96 minutes. At this time, the MRI imaging sequence was performed using 3D Time-Of-Flight Gradient Echo (3D TOF GRE) to acquire images in three dimensions: axial, sagittal, and coronal. The imaging resolution was approximately 0.3 mm. 3 Let's call it voxel.

[0325] 2-5. MR Lymphangiography contrast effect analysis After maximum intensity projection (MIP) of the MRI images taken before contrast injection and those taken at 16-minute intervals for 96 minutes after contrast injection, lymphatic vessels / blood vessels that appear with brighter signals after injection compared to before injection are identified, and the overall quality of the images, including vascular breaks, is evaluated on a four-point scale (-, +, ++, +++). The occurrence of dermal backflow and lymph node congestion is also assessed.

[0326] 2-6. Method for confirming contrast agent absorption at intradermal injection sites 24 hours after the injection of the contrast agent, MRI images of the injection site are taken using 3D Time-Of-Flight Gradient Echo (3D TOF GRE) and compared with the MRI images taken before the injection to confirm whether the contrast agent has been absorbed.

[0327] In the case of INV-001, lymphangiography began 16 minutes after administration and was observed for 64 minutes, demonstrating excellent lymphangiography efficacy (Figure 9). It was also confirmed that minute lymphatic vessels on the order of 300 μm could be observed.

[0328] 2-7. Venous contamination analysis The next day after the imaging was completed, the animals were euthanized and hair was removed. Methylene blue, a dye that selectively stains lymphatic vessels, was then injected intradermally to confirm the anatomy of the lymphatic vessels. The images were then compared with the MRI images to analyze whether or not there was venous contamination.

[0329] When methylene blue was injected intradermally into the lower limb of a rat, the location of lymphatic vessels could be accurately confirmed, and venous contamination could be confirmed by comparing it with MRI images (Figure 10, left side). When comparing the molecular size of INV-001 with that of the existing contrast agent Dotarem (molecular weight 753.9 g / mol, less than 1 nm), INV-001 selectively imaged only lymphatic vessels, while Dotarem showed more visibility of veins than lymphatic vessels, confirming the phenomenon of venous contamination (Figure 10, right side). INV-001, with a hydrated diameter of 3.9 nm, is larger than the 2 nm known to allow venous penetration, confirming that it selectively imaged only lymphatic vessels without venous contamination.

[0330] 2-8. Method for confirming excretion of intradermally injected contrast agent (1) After the contrast agent was injected into the animal, the presence or absence of a contrast effect at the injection site was confirmed 24 hours later. INV-00 was administered intradermally to the lower limb of a rat at a concentration of 20 mM, and MRI was performed 24 hours later. No T1 signal due to the contrast agent was observed at the injection site, confirming that it had all been absorbed into the lymphatic vessels and excreted (Figure 11).

[0331] Example 3. Efficacy analysis of magnetic resonance lymphangiography using INV-001 in healthy beagle dogs The purpose of this example was to verify the efficacy of INV-001 in visualizing lymphatic vessels without venous contamination, and to determine the optimal administration conditions for INV-001 for MRL in beagle dogs.

[0332] 3-1. Animal and Experimental Designations All animal experiments were conducted in accordance with the ethical guidelines and regulations for animal experiments set forth by the Animal Experiment Ethics Committee (IACUC) of the Daegu Gyeongbuk Advanced Medical Industry Foundation (IACUC No. KMEDI-23050401). Furthermore, all procedures in this study complied with the Animal Protection Act and the Guidelines for the Care and Use of Laboratory Animals of the Republic of Korea. Healthy, 2-year-old male beagles (n = 3) weighing 9.3–10.05 kg were used in this study. After overnight fasting (more than 12 hours), the beagles were pre-anesthetized intramuscularly with 0.05 mg / kg atropine and 2 mg / kg xylazine. An intravenous tube was inserted into a hind leg vein, and 2–3 mg / kg of alphaxalone was administered. Once each beagle reached an appropriate state of anesthesia, it was intubated with an appropriately sized intraperitoneal tube. The beagle was then placed on a table and connected to an anesthesia machine and a ventilator (Figure 18). General anesthesia was maintained with inhaled 1–2% isoflurane using a precision vaporizer. INV-001 was injected intradermally into four dorsal web spaces of each hind paw using a small syringe (Figure 19). Each dorsal web space injection site had to be massaged for approximately 2 minutes after INV-001 administration to promote lymphatic absorption.

[0333] First, to determine the optimal concentration and injection volume of INV-001 for MRL in healthy beagles, we conducted a dose-finding study using various concentrations (7.5, 15, 30 mM) and doses (0.028, 0.056, 0.112 mg Fe / kg). Based on qualitative visualization score analysis of lymphatic vessels and lymph nodes, an adaptive optimal dose-finding study design was used to increase the INV-001 administration concentration and dose from 7.5 mM and 0.028 mg Fe / kg (first-order) to 15 mM and 0.056 mg Fe / kg (sixth-order), respectively (Figure 12). In the next step, we conducted a reproducibility study (15 mM concentration) for the determined optimal doses (0.056 and 0.112 mg Fe / kg). Finally, we conducted an excretion study of INV-001 in three healthy beagles. The excretion study utilized T1, T2, and T2* relaxation time measurements to determine the presence of INV-001 in the body at various concentrations and doses (15 mM, 0.056 mg Fe / kg, 30 mM, 0.028 mg Fe / kg, 30 mM, 0.056 mg Fe / kg) within 48 hours after administration.

[0334] 3-2. MRL Acquisition and Scanning Parameters All MRL images were acquired with healthy beagles in a recumbent position using a 3.0 Tesla (T) whole-body clinical magnetic resonance imaging scanner (MAGNETOM Skyra, Siemens Healthcare, Erlangen, Germany, maximum gradient strength 45 mT / m, slew rate 200 T / m / s). MRL images were acquired before and after contrast administration. Two 18-channel body coils (Siemens Healthcare) with 18 integrated preamplifiers were used to visualize the extensive lower lymphatic system from the pelvis to the toes. Additionally, a dual-echo three-dimensional (3D) volumetrically interpolated breath-hold examination (VIBE) sequence with fat suppression (Dixon technique) was used to acquire T1-weighted images in a coronal orientation with two stacks. The scan parameters used were: field of view = 300 × 300 × 115 mm, repetition time (TR) = 6.06 ms, echo time (TE) = 2.46 / 3.69 ms, voxel size = 0.8 × 0.8 × 0.8 mm, acquisition matrix = 384 × 307, acceleration factor = 2, flip angle = 20°, averaging = 1, slice thickness / slice spacing = 0.8 / 0 mm. A detailed summary of the imaging parameters used for MRL is shown in Table 3. After contrast administration, MRL images were performed over five periods (30, 40, 50, 60, and 70 min) using a fat-suppressed tubular 3D VIBE sequence, except for the 7.5 mM and 0.028 mg Fe / kg doses. With the 7.5 mM and 0.028 mg Fe / kg doses, lymphatic vessels, except for the popliteal lymph nodes, were not visualized. Therefore, no further MRL images could be acquired after 50 min. Additionally, T1, T2, and T2* relaxation times were acquired using variable TR, turbo spin echo (TSE) with multiple echoes, and gradient with multiple echo sequences, respectively. The scan parameters used are shown in Tables 2 and 3.

[0335] [Table 2]

[0336] [Table 3]

[0337] a: A parallel imaging technique known as generalized autocorrected partial parallel acquisition (GRAPPA) was used (acceleration factor 2). MR lymphangiograms were obtained in two stacks to visualize the extensive inferior lymphatics. VIBE: volumetric interpolated breath-hold examination, TR: repetition time, TE: echo time, GRE: gradient echo, FOV: field of view.

[0338] 3-3. Quantitative analysis of image quality Quantitative evaluation of contrast enhancement of lymph nodes and lymphatic vessels was performed using the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) (Figure 13). A single reader calculated SNR and CNR values ​​from Image J (Beddess, MD, USA, http: / / rsbweb.nih.gov / ij / ) using the following formula:

[0339] SNR LN = SI LN / Mean SD background CNR LN = (SI LN -SI muscle ) / Mean SD background SNR LV = SI LV / Mean SD background CNR LV = (SI LV -SI muscle ) / Mean SD background where SI LN , S.I. LV , S.I. muscle , and S.D. backgroundThe standard deviations of the signal intensities of the popliteal lymph node, lymphatic vessel, muscle, and background are shown. The T1, T2, and T2* relaxation times of the acquired images were calculated using MATLAB (R2016b, MathWorks, Natick, MA, USA).

[0340] 3-4. Qualitative analysis of image quality Image quality assessment related to lymph node and lymphatic vessel visualization was performed independently by two radiology specialists using visual grading analysis with qualitative scoring criteria to identify optimal concentrations and dosages. The criteria used were 1) venous contamination, 2) skin reflux, 3) lymph node congestion, and 4) lymph node and lymphatic vessel visualization scores. A detailed summary of the qualitative scoring criteria is provided in Table 4.

[0341] [Table 4]

[0342] This scoring system (without a 1) was used so that higher scores reflect better image quality.

[0343] 3-5. statistical analysis Interobserver agreement for qualitative assessment was assessed using a weighted kappa score. The strength of interobserver agreement using the weighted kappa score was assessed as follows: ≤0.2, poor; 0.21-0.40, fair; 0.41-0.60, fair; 0.61-0.80, good; and 0.81-1.00, excellent. The Kolmogorov-Smirnov test was used to confirm normal distribution of relaxation times. T1, T2, and T2* relaxation time values ​​as a function of anatomical lesion were compared using paired Student's t-tests. Statistical analyses were performed using IBM SPSS Statistics for Windows / Macintosh, v. 26.0 (IBM Corp., Armonk, NY, USA). For all statistical analyses, a two-sided p<0.05 level was considered to indicate a statistically significant difference.

[0344] [result] 1. Dose-finding study The results of qualitative lymphatic scoring analysis for the adaptive optimal dose detection study under various administration conditions of 0.028, 0.056, and 0.112 mg Fe / kg at various concentrations (7.5, 15, and 30 mM) are shown in Table 5. No venous contamination was observed under any of the test conditions. Qualitative image analysis results showed that the 0.028 mg Fe / kg dose at concentrations of 7.5, 15, and 30 mM yielded relatively low total scores of 2.5, 4.5, and 3.5, respectively (Figure 14a and Table 5). The 0.056 mg Fe / kg dose and concentrations of 15 mM and 30 mM yielded total scores of 6 and 5, respectively. Because the total score was higher at 15 mM than at 30 mM for the same dose of 0.056 mg Fe / kg, a higher dose of 0.112 mg Fe / kg was tested, resulting in a total score of 6 (Figure 14b and Table 5). Quantitative analysis showed that the lymphatic vessels and nodes showed the best contrast enhancement at 15 mM and 0.056 and 0.112 mg Fe / kg doses. Specifically, the SNR was 0.112 mg Fe / kg at 15 mM. LN , SNR LV , CNR LN , and CNR LV showed the highest values ​​of 295.62 ± 27.08, 233.14 ± 20.31, 216.22 ± 20.21, and 153.16 ± 13.61, respectively (Figure 15). Based on these quantitative and qualitative analyses, the dose conditions of 0.056 and 0.112 mg Fe / kg at a concentration of 15 mM were selected for a dose expansion study to evaluate reproducibility.

[0345] [Table 5]

[0346] 2. Reproducibility study The results of the dose expansion study for reproducibility assessment are shown in Table 5. All acquired MRL images clearly show the visibility of lymphatic vessels and lymph nodes without venous contamination. In qualitative analysis, the 0.056 and 0.112 mg Fe / kg doses at 15 mM concentration received total scores of 5.33 and 5.66, respectively (Table 6). Overall, the qualitative analysis results of the two groups show good visualization results of lymphatic vessels and lymph nodes. No venous contamination or skin reflux was observed in all test conditions (Figure 16). In quantitative analysis measured 30 minutes after administration, the SNR LN , SNR LV , CNR LN , and CNR LV The 0.056 mg Fe / kg group showed maximum values ​​of 178.12 ± 32.84, 133.76 ± 30.33, 119.96 ± 43.40, and 83.94 ± 19.43, respectively (Figure 17). The 0.112 mg Fe / kg group recorded values ​​of 257.34 ± 61.10, 195.44 ± 45.26, 199.37 ± 61.58, and 138.32 ± 48.03. These values ​​also showed a tendency to decrease gradually up to 70 minutes, with the decrease rate ranging from 7.3 to 22.8%, confirming sustained visualization of lymphatic vessels and lymph nodes. Compared to the 0.056 mg Fe / kg group, all test results for the 0.112 mg Fe / kg group showed higher values ​​for lymphatic vessels and lymph nodes. These results indicate that INV-001 more effectively visualized lymphatic vessels and lymph nodes in a dose-dependent manner. Quantitative and qualitative analysis showed that at doses of 0.056 and 0.112 mg Fe / kg and a concentration of 15 mM, lymphatic vessels and lymph nodes could be visualized without venous contamination, with excellent reproducibility. Therefore, it can be concluded that a dose higher than 0.056 mg Fe / kg at a concentration of 15 mM INV-001 is sufficient for the MRL in beagles.

[0347] [Table 6]

[0348] 3. Excretion research To confirm the excretion of INV-001, the T1, T2, and T2* relaxation times in the liver and kidney were analyzed at time 0 and 48 hours after administration (Table 7). INV-001 was administered at a concentration of 15 mM at a dose of 0.056 mg Fe / kg. Compared to pre-injection (time 0), the T1, T2, and T2* relaxation times measured in the liver and kidney at 48 hours showed no statistically significant differences, except for the hepatic T2* relaxation time (p<0.05) (Figure 20). There was also no statistically significant difference in the T2* relaxation time measured in the liver and kidney (p>0.05). Furthermore, INV-001 did not remain at the injection site (Figure 21). Therefore, it was determined that INV-001 was effectively excreted from the body within 48 hours.

[0349] [Table 7]

[0350] Example 4: 4-1. Synthesis of nanostructures for intra-articular injection 180 μmol of dextran (average molecular weight 10,000 Da) was dissolved in 9 mL of distilled water, and epichlorohydrin and NaOH were added. Ethylenediamine was then added, and the mixture was stirred at room temperature (RT) for 24 hours. After 24 hours of succinylation at room temperature, the dextran core was purified by dialysis using a 15 kD molecular weight cutoff (MWCO) filter. An excess of iron chloride hexahydrate solution was added to the dextran core solution. The pH was adjusted to 8 using 1 M NaOH, and the mixture was incubated at room temperature for 1 hour. The nanostructure (code name: INV-002) was synthesized by dialysis. The iron concentration at maximum concentration was 1.6 mg / mL. The viscosity was 7.1 cP (Figure 8).

[0351] The nanostructures synthesized in Examples 2-1 and 4-1 were visually observed as yellow or yellowish-brown transparent solutions, and the iron and dextran contents were analyzed by inductively coupled plasma (ICP) and the phenol-sulfuric acid method, respectively, and the ratio was determined to be approximately 3:100 by mass. The number of functional groups derived from the crosslinker was confirmed by ortho-phthalaldehyde assay and elemental analysis after the crosslinking reaction. The ratio of crosslinked monosaccharides to the total number of monosaccharides in the polysaccharide crosslinked colloidal particles was controlled to 2% to 60%, and the crosslinker substitution ratio was 10% to 50% of the number of dextran functional groups, of which 20% to 50% did not participate in crosslinking and had their ends exposed to the outside. When ortho-phthalaldehyde quantification was performed again after iron ion binding, crosslinker-derived functional groups were detected, confirming that some of the crosslinker-derived functional groups were bound to iron ions and the remaining functional groups were exposed in an unbound form. Dynamic light scattering analysis of the synthesized nanostructures confirmed that the hydrodynamic diameters were uniformly distributed within the renal filtration cutoff of 8 nm, and the charge ranged from -20 mV to 0 mV. Gel permeation chromatography measured the average molecular weights of INV-001 and INV-002 to be approximately 15 kD and 30 kDa, respectively. Given that the nanostructures were synthesized using dextran with average molecular weights of approximately 5,000 Da and 10,000 Da, respectively, it was determined that two to three dextran molecules were crosslinked.

[0352] 4-2. T1-MRI contrast effect of nanostructured material INV-002 for intra-articular injection To confirm whether INV-002 of Example 4-1 can be used as a T1-MRI contrast agent for magnetic resonance imaging arthrography (MR arthrography), a test was conducted to determine whether the T1-MRI contrast effect was maintained even after mixing with an iodinated X-ray contrast agent.

[0353] For testing, a mixture of INV-002 (Example 4-1) and iopamidol, a representative iodinated X-ray contrast agent, was prepared. The INV-002-iopamidol mixture generally exhibited stronger T1-MRI contrast effects than INV-002 at the same concentration. For example, a 1:1 mixture of INV-002 and iopamidol exhibited approximately twice the T1-MRI contrast effects of INV-002 at the same concentration. These results confirm that INV-002 (Example 4-1) maintains its strong T1-MRI contrast effects even when mixed with an iodinated X-ray contrast agent, making it effective for use in magnetic resonance imaging arthrography.

[0354] 4-3. Magnetic resonance imaging arthrography using nanostructured material INV-002 for intra-articular injection Male Sprague-Dawley rats aged 7 weeks or older were anesthetized using a mixture of oxygen and isoflurane, and then INV-002 of Example 4-1 was injected into the knee joint. T1-weighted MRI images were then taken using a fast-spin echo sequence.

[0355] As shown in Figures 22(a)-(d), the knee joint cavity of animals injected with INV-002 appears significantly brighter on MRI images than the knee joint cavity of animals injected with INV-002. This change in signal intensity can also be quantitatively confirmed, as shown in Figure 22(e). For example, the T1 signal intensity of the synovial fluid increased from 4,900 (before injection) to 21,000 (after injection). The CNR of intra-articular tissues, including the crescentic cartilage, capsule, cruciate, bone, and fat regions, was then analyzed (Figure 22(f)). Here, CNR is calculated by dividing the difference in mean signal intensity between the synovial fluid and each anatomical region by the standard deviation of the background signal intensity. After INV-002 injection, the CNR of the crescentic cartilage, capsule, cruciate, bone, and fat regions increased by 10, 8.5, 18, 14, 12, and 4-fold, respectively, compared to before injection.

[0356] The contrast effect of INV-002 was not only stronger but also longer-lasting than that of gadolinium contrast agents (e.g., Dotarem). For quantitative analysis of the contrast effect, SNR was measured at two locations within the joint cavity (orange arrows) and the average values ​​were plotted over time. INV-002 and Dotarem exhibited the highest SNR at 0.25 hours after injection, where the SNR of INV-002 was approximately two-fold higher than that of Dotarem (Figures 23(a) and 23(b)). The SNR of Dotarem decreased to pre-injection levels within 0.5 hours, whereas INV-002 maintained a significant SNR for up to 6 hours after injection, with no contrast enhancement observed from 9 hours after injection (Figure 23(c)).

[0357] These results indicate that INV-002 of Example 4 exhibits intra-articular contrast enhancement effect and is useful for identifying intra-articular anatomical structures. It was analyzed that the contrast effect lasts longer than that of Dotarem, providing useful opportunities for high-resolution imaging, repeated imaging, and re-imaging in the event of imaging failure (Figure 23).

[0358] 4-4. Excretion of nanostructured product INV-002 for intra-articular injection As shown in the results of Example 4-3, no contrast enhancement was observed 9 hours after INV-002 was injected into the knee joint cavity of rats, indicating that INV-002 was absorbed and excreted from the joint cavity within 9 hours. To more accurately analyze the excretion pathway, urine was collected after INV-002 was injected into the knee joint cavity of Sprague-Dawley rats aged 7 weeks or older. The iron content in urine collected from 0 to 24, 24 to 48, and 48 to 72 hours was analyzed using ICP. In the control group, saline was injected, and urine was collected at the same time points and in the same manner, and the iron content was analyzed.

[0359] As a result, as shown in Figure 24(a), the iron content in urine collected from male and female rats injected with INV-002 was statistically significantly different from that of the saline-injected control group. In particular, the total amount of iron in urine was approximately 0.018 mg, which was analyzed to be identical to the amount of injected iron within the margin of error. This indicates that INV-002 injected into the joint cavity is largely excreted from the body through urine excretion within 24 hours after injection. As shown in Figure 24(b), the color of urine samples collected after INV-002 injection was analyzed to be a darker brown color compared to the saline-injected control group due to the color of INV-002.

[0360] To confirm whether INV-002 injected into the joint cavity was completely eliminated, microscopic analysis was performed using immunohistochemical staining (Perls Prussian blue iron staining), which detects even trace amounts of iron. Joint tissue from INV-002-injected experimental animals was collected, fixed in 10% neutral buffered formalin, stained with Perls Prussian blue iron, and then examined under a digital microscope. As a result, as shown in Figure 25, no iron accumulation was observed in the INV-002-injected group (Figure 25(a)) or the saline-injected control group (Figure 25(b)).

[0361] From the above results, it was analyzed that INV-002 injected into the joint cavity was excreted in the urine without accumulating in the joint cavity.

[0362] 4-5. Imaging effect and excretion of nanostructured substance INV-002 injected into the spinal cavity Following the results of Example 4-3, which confirmed that INV-002 injected into the joint cavity was expelled from the joint cavity, an experiment was conducted to confirm whether INV-002 exhibited a contrast effect when injected into the spinal cavity and whether it could be expelled. Sprague-Dawley rats aged 7 weeks or older were anesthetized with a mixture of oxygen and isoflurane, and INV-002 (Example 4-1) was injected into the spinal cavity. T1-weighted images were then taken using a fast-spin echo sequence.

[0363] As shown in Figure 26, the spinal cord cavity of animals injected with INV-002 was observed as a bright signal in T1-MRI images. Immediately after injection, the spinal cord cavity became brighter compared to pre-injection, reaching its maximum brightness at 30 and 60 minutes after injection. The brightness decreased at 90 minutes after injection, and by 120 minutes after injection, it had decreased to a level similar to pre-injection.

[0364] From the above, it was analyzed that the nanostructure INV-002 injected into the spinal cavity was expelled from the spinal cavity.

[0365] 4-6. Single-dose safety study of the nanostructured substance INV-002 for intra-articular injection To evaluate the local intra-articular toxicity of INV-002, a single dose of INV-002 was administered intra-articularly to Sprague-Dawley rats and Beagle dogs, followed by a two-week observation period. The INV-002 concentration used in the local toxicity study was 1.6 Fe mg / mL, the highest concentration possible (Figure 27). This is approximately 11-fold higher than the 0.14 Fe mg / mL concentration of the drug planned for clinical trials. The maximum intra-articular volume (0.1 mL for rats and 1 mL for Beagle dogs) was administered. The amount injected into rats was 0.16 Fe mg / head, and that of Beagle dogs was 1.6 Fe mg / head.

[0366] As a result, no abnormalities were observed, and as shown in Figure 27, no difference in body weight patterns was observed between the INV-002-injected group and the control group. Therefore, the NOAEL (No Observed Adverse Effect Level) was determined to be 0.160 Fe mg / head and 1.60 Fe mg / head, respectively, which are 23 and 6 times the planned clinical dose, indicating excellent safety.

[0367] 4-7. Single-dose blood chemistry and histopathological studies of the nanostructured product INV-002 for intra-articular injection Since INV-002 in Example 4-1 was shown to be absorbed into the circulatory system and excreted via the renal elimination pathway, blood chemistry tests were performed to evaluate liver function (alanine transaminase, ALT; aspartate transaminase, AST; alkaline phosphatase, ALP; gamma-glutamyltransferase, GGT) and kidney function (blood urea nitrogen, BUN; creatinine, CR). For blood chemistry analysis, blood was collected from the abdominal aorta and serum was separated by centrifugation. ALT, AST, ALP, GGT, BUN, and CR values ​​were analyzed using an automated biochemistry analyzer.

[0368] For histopathological examination, each organ was fixed in 10% neutral buffered formalin and then processed for hematoxylin and eosin staining according to the protocol provided by the manufacturer. Tissue sections were counterstained with eosin for 1 minute and then analyzed under a digital microscope.

[0369] As a result, as shown in Figure 28, all blood chemistry test items were within the normal range, and the histopathological results showed no pathological abnormalities or lesions, indicating that INV-002 has excellent biocompatibility.

[0370] No abnormal changes in cell structure (e.g., collapse, distortion, or dilation) were observed.

[0371] No organs showed hemorrhage, inflammation or necrosis, indicating the non-toxicity of INV-002. Blood chemistry analysis confirmed normal kidney and liver function in INV-002 injected rats.

[0372] Example 5: Shoulder MR arthrography using an iron-based positive T1 contrast agent (NEMO-103)

[0373] 5-1. Preparation of dextran-based T1-MRI contrast agent NEMO-103 A dextran crosslinker was synthesized by mixing 20 mM dextran T10 in water with sodium hydroxide solution and epichlorohydrin, followed by the addition of ethylenediamine. The synthesized dextran crosslinker was purified using a 10 kD molecular weight cutoff (MWCO) filter. The terminal amines of the dextran crosslinker were converted to carboxyl functional groups using succinic anhydride. After purification using a 10 kD molecular weight cutoff (MWCO) filter, the carboxyl functionalized dextran crosslinker was reacted with iron chloride solution for 1 hour and then purified using a 10 kD molecular weight cutoff (MWCO) filter to prepare the dextran crosslinker-based T1-MRI contrast agent NEMO-103 (Figure 29a).

[0374] 5-2. Phantom study The NEMO-103 phantom was imaged using 3T MRI, and the T1-weighted images showed bright T1 signals similar to those of the dota-lemma (Fig. 29b, c). The measured r1 and r2 values ​​of NEMO-103 were 2.0mM, respectively. -1 s -1 and 2.3 mM -1 s -1(Fig. 29d, e). The r2 / r1 ratio, an important parameter of a T1-MRI contrast agent, was calculated to be 1.15. Considering that the closer the r2 / r1 ratio is to 1, the more ideal the T1-MRI contrast agent, NEMO-103, which exhibits a value of 1.15, is an excellent T1-MRI contrast agent. For comparison, the r1, r2, and r2 / r1 ratios of Dotarem, which were measured, were 4.7 mM, respectively. -1 s -1 , 5.2mM -1 s -1 , was observed to be 1.11.

[0375] 5-3. Image Quality Assessment Figures 30 and 31 show the phase classification and image quality assessment method for a clinical comparison study of NEMO-103, a dextran crosslinked-based T1-MRI contrast agent, and Dotarem, a representative gadolinium-based contrast agent (GBCA) for clinical use in T1-MRI. Phase I represents magnetic resonance arthrography (MR arthrography) images taken up to 30 minutes after contrast administration. Phase II represents MRA images taken from 30 to 60 minutes after contrast administration. Phase 3 represents MRA images taken between 110 and 130 minutes after contrast administration. In Comparison 1, there was no difference in quantitative or qualitative quality between NEMO-103-based MRA images in clinical Phase I and Phase II (Figure 32). In comparison 2, when comparing NEMO-103-based and GBCA-based MRA images taken in Phase I, no difference in CNR was observed, but a significant difference was found in the degree of distension (p<0.05, Figure 33). As a result, NEMO-103-based MRA images received higher scores for overall image quality. Regarding distension, there was no significant difference in the degree of posterior joint pocket distension between the two groups. However, NEMO-103-based MRA images showed better results in both the lower joint pocket and axillary pocket distension. In comparison 3, comparing NEMO-103-based and GBCA-based MRA images taken in Phase II, a more significant difference was observed (Figure 34). CNR was significantly higher in NEMO-103-based MRA images than in GBCA-based MRA images. Furthermore, even more significant differences were observed in the degree of lower capsule distension and axillary pocket distension. As a result, NEMO-103-based MRA images showed better results for both lower joint pocket swelling and axillary pocket swelling.

[0376] 5-4. Visual Turing Test (VTT) In the VTT for comparison 1, radiologists were assigned to distinguish between Phase I and Phase II NEMO-103-based MRA images. The combined accuracy of the eight testers was 46.8% (146 / 312), which was not statistically significant compared with random guessing (50.0%, 156 / 312) (p = 0.423). In the VTT for comparison 2, radiologists were assigned to distinguish between Phase I NEMO-103-based MRA images and Phase I GBCA-based MRA images. The combined accuracy of the 32 testers was 53.3% (624 / 1170), which was not statistically significant compared with random guessing (50.0%, 585 / 1170) (p = 0.107).

[0377] [Discussion] Recently, concerns about the safety of GBCAs have been raised, leading to research into iron (Fe)-based alternatives. This example evaluates the efficacy of NEMO-103 by comparing CNR and MRA image quality with those of gadolinium-enhanced contrast agents (GBCAs). In shoulder MR arthrography, NEMO-103 (0.035 mg Fe / kg) and GBCA (0.037 mg Gd / kg) were directly injected into the joint, and CNR and visual Turing test (VTT) results showed nearly identical image quality at 30 minutes (Comparison 2). Both agents demonstrated clearness, contrast, and effective depiction of structures such as the rotator cuff muscles, labral structures, and surrounding cartilage. NEMO-103 also demonstrated improved CNR 120 minutes after injection.

[0378] In this example, the quality of MRA images taken at 30 minutes (phase 1) and 60 minutes (phase 2) was indistinguishable using a visual Turing test, suggesting that imaging time after injection may be extended. This is beneficial for the operational efficiency of MR imaging facilities in actual clinical practice. According to current reports, microparticle contrast agents with a hydrodynamic diameter of 3 nm are excreted from the joint cavity only via lymphatic vessels and not via veins due to their large size. NEMO-103, with an even larger hydrodynamic size (approximately 4.0 nm), can only be excreted via lymphatic vessels, resulting in a longer residence time in the joint cavity. In contrast, GBCAs (<1 nm) are rapidly excreted via lymphatic vessels and veins, limiting imaging time. However, MRA images taken 24 hours after administration confirmed that NEMO-103 had been completely eliminated from the joint cavity, with no detectable residue.

[0379] On the other hand, there was no significant difference in posterior pocket expansion between NEMO-103 and GBCA-based MRA images. This is presumably due to the patient's position during shoulder MRA. When lying down, synovial fluid tends to move toward the posterior pocket, leaving relatively little residual fluid in structures such as the subscapularis, axillary pocket, and subscapularis. Over time, as the contrast agent is reabsorbed, this difference becomes more apparent with GBCA. As a result, GBCA showed a relatively higher incidence of axillary pocket disappearance compared to NEMO-103. Furthermore, the anterior aspect of the humeral joint contains important structures, such as the subscapularis muscle and the anterior inferior glenoid frenulum. Because there are many structures to evaluate in this area, synovial fluid absorption and fluid movement due to lying down can actually affect the visibility of anterior lesions.

[0380] Unlike the long-established use of GBCAs, the novel NEMO-103 requires safety evaluation. According to the clinical study report, three (9.4%) of 32 subjects receiving the NEMO-103-based MRA experienced treatment-emergent adverse events (TEAEs), including two cases of local injection site reactions (myalgia) and one case of hypertension. These adverse events were deemed "not related to the investigational drug" in terms of causality, and all subjects recovered without intervention. Furthermore, no drug-related adverse events (ADRs), serious adverse events (SAEs), or serious adverse events occurred, and no related dropouts occurred. Evaluation of NEMO-103 residues in the liver and spleen 24 hours after administration confirmed complete clearance in all 30 subjects (100%). The difference in rSI between pre-contrast and 24-hour delayed abdominal MRI was positive in both cases (liver 13.2±4.0, spleen 5.5±0.9). Two patients did not have 24-hour delayed imaging, including liver and spleen imaging.

[0381] Another benefit of NEMO-103 is that it eliminates the need for existing off-label use. Existing GBCA-based MRA requires the GBCA to be diluted approximately 1:200, which increases the risk of infection and contamination and hinders standardization of contrast agent concentration. However, NEMO-103 is provided as a vial product already diluted for arthrography, making it relatively free from issues such as infection, contamination, and heterogeneous contrast agent concentration.

[0382] As a retrospective study with a limited number of patients, this study has several limitations. First, because it did not include patients who underwent arthrography, we were unable to evaluate the diagnostic accuracy of NEMO-103-based and GBCA-based shoulder MRA for each lesion. However, by emphasizing the role of intra-articular contrast agents, such as improving contrast with surrounding structures and joint cavity expansion, we are able to observe meaningful differences over time between NEMO-103- and GBCA-based MRA images. Furthermore, a visual Turing test involving many testers showed that NEMO-103 demonstrated superior image quality to GBCA in various aspects. Clinical trials can be conducted using surgical records as a standard to evaluate the diagnostic accuracy of the two contrast agents in patients who require future surgery. Second, this study did not evaluate both contrast agents in the same patient. Individual differences in contrast-induced joint cavity expansion and contrast reabsorption may exist. However, performing two MRAs using different contrast agents within a short period of time on the same patient is a challenging study design. To overcome this, we carefully examined the time from contrast injection to MRI and compared the data using phase matching. Third, we noted that the GBCA group was significantly older than the NEMO-103 group. Although this has not been conclusively proven, there may be potential differences in contrast absorption rates depending on age. However, given that synovial fluid tends to increase with more severe degeneration and symptoms such as pain, it is possible that the GBCA group, which was composed of patients, actually had a greater amount of synovial fluid than the relatively young volunteer-based NEMO-103 group.

[0383] In conclusion, NEMO-103-based MR shoulder arthrography is at least interchangeable with GBCA-based MR shoulder arthrography in terms of CNR, expansion, image quality, and Turing test results, and suggests that the resistance to contrast reabsorption may improve over time, potentially allowing for an extended acquisition period.

[0384] Example 6 6-1. Synthesis of C-DNP-3 cross-linked with 3 kDa dextran 1 g of dextran (molecular weight 3 kDa) was dissolved in 4.2 mL of distilled water, followed by the addition of 8.3 mL of NaOH solution. 3.3 mL of epichlorohydrin was added and stirred. 7 mL of diethylenetriamine (DETA) was added and the mixture was further stirred for 24 hours to synthesize C-DNP-3 crosslinked with 3 kDa dextran. After purification by ultrafiltration, the hydrodynamic size measured by DLS was 3 nm.

[0385] 6-2. Synthesis of C-DNP-5 cross-linked with 5 kDa dextran C-DNP-5 crosslinked with 5 kDa dextran was synthesized in the same manner as in Example 6-1, except that dextran (molecular weight 5 kDa) was used instead of dextran (molecular weight 3 kDa). After purification by ultrafiltration, the hydrodynamic size measured by DLS was 4 nm.

[0386] 6-3. Synthesis of C-DNP-10 cross-linked with 10 kDa dextran C-DNP-10 cross-linked with 10 kDa dextran was synthesized in the same manner as in Example 6-1, except that dextran (molecular weight 10 kDa) was used instead of dextran (molecular weight 3 kDa). After purification by ultrafiltration, the hydrodynamic size measured by DLS was 5 nm.

[0387] 6-4. Synthesis of C-CMDNP-10 cross-linked with 10 kDa carboxymethyl dextran (CM dextran) C-CMDNP-10 crosslinked with 10 kDa CM dextran was synthesized using the same method as in Example 6-1, except that CM dextran (molecular weight 10 kDa) was used instead of dextran (molecular weight 3 kDa). After purification by ultrafiltration, the hydrodynamic size measured by DLS was 5 nm.

[0388] 6-5. Substitution of functional groups exposed on the surface of C-DNP or C-CMDNP 6-5-1. Amine group: The functional group exposed on the surface of the C-DNP or C-CMDNP synthesized in Examples 6-1 to 6-4 is an amine group.

[0389] 6-5-2. Carboxyl group: 30 mg succinyl anhydride (SA) was added to 10 mL of the C-DNP or C-CMDNP synthesized in Examples 6-1 to 6-4, and the mixture was stirred for 12 hours. The final C-DNP or C-CMDNP was purified by ultrafiltration.

[0390] 6-5-3. Thiol group: N-succinimidyl S-acetylthioacetate was added to 10 mL of C-DNP or C-CMDNP synthesized in Examples 6-1 to 6-4, and the mixture was stirred for 12 hours. The final C-DNP or C-CMDNP was purified by ultrafiltration.

[0391] 6-5-4. Hydroxide group: Nitrous acid was added to 10 mL of the C-DNP or C-CMDNP synthesized in Examples 6-1 to 6-4, and the mixture was stirred for 12 hours. The final C-DNP or C-CMDNP was purified by ultrafiltration.

[0392] 6-6. Surface charge control of C-DNP / C-CMDNP When 30 mg of SA was added to 10 mL of C-DNP synthesized in Examples 6-1 to 6-3 using Example 6-5-2, the surface charge was -3 mV. When 50 mg of SA was added, the surface charge was -20 mV. When no SA was added, the surface charge was +5 mV. Experiments using C-DNP-3, C-DNP-5, and C-DNP-10 showed identical results for the surface charge depending on the amount of SA added, all under the same functional group substitution conditions. When 30 mg of SA was added to 10 mL of C-CMDNP-10 synthesized in Example 6-4, which had a surface charge of +5 mV, using Example 6-5-2, the surface charge was -4 mV. As a representative example, Figure 35 shows the surface charge of C-DNP-10 depending on the amount of SA added. As such, the surface charge becomes more negative as the amount of SA added increases, allowing for adjustment of the surface charge.

[0393] 6-7. Inorganic nanoparticle surface coating using C-DNP / C-CMDNP Ten mg of 4 nm iron oxide nanoparticles (Figure 36(a) and (b)) were placed in tetramethyl ammonium hydroxide (TMAOH) and stirred. The supernatant was discarded by magnetic decantation, and the nanoparticles were washed twice with 10 mL of hexane and acetone. The washed iron oxide nanoparticles were mixed with 100 mg of carboxyl-substituted C-DNP-3, C-DNP-5, C-DNP-10, and C-CMDNP-10 prepared in Example 6-5-2 and stirred for 12 hours. C-DNP / C-CMDNP-coated iron oxide nanoparticles (IONP@C-DNP-3, IONP@C-DNP-5, IONP@C-DNP-10, and IONP@C-CMDNP-10) were purified by ultrafiltration.

[0394] 6-8. Hydrodynamic size of inorganic nanoparticles coated with C-DNP / C-CMDNP The hydrodynamic sizes of IONP@C-DNP-3, IONP@C-DNP-5, and IONP@C-DNP-10 prepared in Examples 6-7 were 7 nm, 9 nm, and 11 nm, respectively (Figure 36(c)). Because the size of the carboxymethyl (CM) group (unit: 1000kJ / s) was small enough not to affect the overall particle size (unit: nanometers), the hydrodynamic size of IONP@C-CMDNP-10 prepared in Examples 6-7 was also 11 nm (Figure 36(d)).

[0395] 6-9. Surface charge of nanoparticles coated with C-DNP with various surface charges Surface coating of 4 nm iron oxide nanoparticles was performed as in Examples 6-7 using C-DNP-3, C-DNP-5, and C-DNP-10, which were prepared in Examples 6-7 and had surface charges adjusted to -20, -3, and +5 mV at pH 7. The surface charges of iron oxide nanoparticles coated with -20 mV C-DNP were -20 mV, -3 mV, and +5 mV, respectively, and those coated with C-DNP were +5 mV. When iron oxide nanoparticles were surface-modified with C-DNP, the surface charge was the same as that of C-DNP, regardless of the surface charge of the iron oxide nanoparticles, indirectly confirming that the iron oxide nanoparticle surface was uniformly coated with C-DNP.

[0396] As a representative example, the surface charge of iron oxide nanoparticles coated with C-DNP-10, with the surface charge adjusted to -20, -3, and +5 mV, is shown in Figure 37. In this way, it is possible to adjust the surface charge of coated iron oxide nanoparticles using C-DNP with adjusted surface charge.

[0397] 6-10. Colloidal stability of iron oxide nanoparticles coated with C-DNP and C-CMDNP Solutions corresponding to physiological conditions (0, 0.15, 0.5, 1.0 M NaCl) and pH (5, 7, 8) were prepared. 0.1 mg of C-DNP- or C-CMDNP-coated iron oxide nanoparticles (IONP@C-DNP-10, IONP@C-CMDNP-10) prepared in Examples 6-7 were added to each solution, and colloidal stability was monitored for 28 days. As shown in Figure 38(a), the C-DNP-10-coated iron oxide nanoparticles of Examples 1-9 remained dispersed for extended periods without precipitation due to aggregation under various physiological conditions (salt, pH). Furthermore, as shown in Figure 38(b), the C-DNP-10-coated iron oxide nanoparticles of Examples 6-6 showed no change in hydrodynamic size over the observation period, confirming their excellent colloidal stability. The C-CMDNP-10 coated iron oxide nanoparticles of Example 6-6 were also confirmed to have excellent colloidal stability without precipitation, as shown in Figure 39(a).

[0398] 6-11. Surface coating of inorganic nanoparticles using non-crosslinked common dextran Ten mg of 4 nm iron oxide nanoparticles were placed in tetramethyl ammonium hydroxide (TMAOH) and stirred. The supernatant was discarded by magnetic decantation, and the nanoparticles were washed twice with 10 mL of hexane and acetone. The washed iron oxide nanoparticles were mixed with 50, 100, 500, 1000, and 2000 mg of uncrosslinked common dextran (molecular weight 10 kDa) and stirred for 12 hours. The common dextran-coated nanoparticles were purified by ultrafiltration. The hydrodynamic sizes of the iron oxide nanoparticles coated with 50, 100, 500, 1000, and 2000 mg of dextran were 160, 85 nm, 49 nm, 26 nm, and 18 nm, respectively. As can be seen from Figure 39(b), the minimum hydrodynamic size of iron oxide nanoparticles achievable with dextran (molecular weight 10 kDa) is 18 nm, and the minimum amount of dextran required for this is 2,000 mg. This is 20 times the amount of C-DNP-10 required for iron oxide coating (100 mg). In other words, when using non-crosslinked general dextran, a 20-fold larger amount than C-DNP-10 is required to ensure the colloidal stability of iron oxide nanoparticles. Furthermore, as shown in Figure 39(a), iron oxide nanoparticles coated with 100 mg of C-DNP-10 did not precipitate in a colloidal stability experiment, whereas when iron oxide was coated with 100 mg of non-crosslinked general dextran, precipitation occurred within 7 days under physiological conditions. Therefore, when C-DNP is used, coating is possible with a 20-fold smaller amount than dextran, and excellent colloidal stability can be ensured.

[0399] This is believed to be due to the compact spherical shape of the dextran crosslinked nanoparticles of the present invention, the ability to form strong coordinate bonds between the functional groups (carboxyl groups or amine groups) derived from the crosslinker and the iron oxide nanoparticles, and the excellent colloidal stability.

[0400] 6-12: Coating inorganic nanoparticles with uncrosslinked CM dextran 10 mg of 4 nm iron oxide nanoparticles were placed in tetramethyl ammonium hydroxide (TMAOH) and stirred. The supernatant was discarded by magnetic decantation, and the nanoparticles were washed twice with 10 mL of hexane and acetone. The washed iron oxide nanoparticles were mixed with 2,000 mg of uncrosslinked CM dextran (molecular weight 10 kDa) and stirred for 12 hours. CM dextran-coated nanoparticles were purified by ultrafiltration. The hydrodynamic size of iron oxide nanoparticles coated with uncrosslinked CM dextran was 18 nm. Furthermore, as shown in Figure 39(a), iron oxide nanoparticles coated with 100 mg of C-CMDNP-10 did not precipitate in a colloidal stability experiment, whereas iron oxide nanoparticles coated with 100 mg of uncrosslinked CM dextran precipitated within 7 days under physiological conditions.

[0401] 6-13. Anti-opsonization effect of C-DNP coated iron oxide nanoparticles Magnetic nanoparticles coated with C-DNP-5, prepared in Examples 6-11 and adjusted to charges of -20, -3, and +5 mV, were mixed with serum proteins (FBS, fetal bovine serum) and left for 10 minutes. The mixture was loaded onto a 1% agarose gel, followed by electrophoresis, and the migration of the nanoparticles was observed by photograph (Figure 40). Figure 40 compares and presents the effectiveness of preventing serum protein binding by band shift and broadening due to the surface charge of C-DNP-5-coated magnetic nanoparticles. Magnetic nanoparticles coated with C-DNP-5, with a surface charge of +5 mV, migrate in the direction of the (-) electric field (Figure 40(a)). In contrast, when serum proteins are mixed with magnetic nanoparticles coated with C-DNP-5, which has a surface charge of +5 mV, the serum proteins bind to the surface of the magnetic nanoparticles, increasing their hydrodynamic size and randomly changing the surface charge. As a result, band broadening is observed and the migration direction also changes (Figure 40(a)). Magnetic nanoparticles coated with C-DNP-5, which has a surface charge of -3 mV, migrate slightly toward the (+) electric field (Figure 40(b)). When this material is mixed with serum proteins, the serum proteins do not bind to the magnetic nanoparticle surface, so no band broadening is observed and the migration direction in the electric field remains the same (Figure 40(b)). Magnetic nanoparticles coated with C-DNP-5, which has a surface charge of -20 mV, migrate significantly toward the (+) electric field (Figure 40(c)). When serum proteins are mixed with magnetic nanoparticles coated with C-DNP-5, which has a surface charge of -20 mV, the serum proteins bind nonspecifically to the surface of the magnetic nanoparticles, increasing their hydrodynamic size and resulting in band broadening (Figure 40(c)).

[0402] As shown in FIG. 40, the electrophoresis results of C-DNP-coated iron oxide nanoparticles confirm the anti-opsonization effect due to the surface charge adjustment of the nanoparticles.

[0403] 6-14. Pharmacokinetic analysis of C-DNP-coated iron oxide nanoparticles by adjusting C-DNP size and surface charge As shown in Figure 41, 4nm iron oxide nanoparticles were coated with various types of C-DNP and injected into animals, and the MRI signal changes over time were observed. Here, 4nm iron oxide exhibits a contrast effect in two different MRI imaging modes, T1 and T2, and this characteristic was utilized in the pharmacokinetic analysis.

[0404] Iron oxide nanoparticles coated with C-DNP-3 (hydrodynamic size: 3 nm) with a surface charge of -3 mV have a hydrodynamic size of 7 nm. Because this material has a hydrodynamic size of less than 8 nm and a surface charge close to 0 mV, it is not phagocytosed by macrophages, and intravenously administered materials can be excreted through the kidney. Therefore, we confirmed the renal excretion of C-DNP-3-coated iron oxide nanoparticles administered to mice using T1-MRI images (Figure 41(a)). Iron oxide nanoparticles coated with C-DNP-10 (hydrodynamic size: 5 nm) with a surface charge of -3 mV have a hydrodynamic size of 11 nm. Because this material has a hydrodynamic size of more than 8 nm and a surface charge close to 0 mV, it is not easily phagocytosed and can remain in blood vessels for long periods of time, making it advantageous for long-term vascular observation (Figure 41(b)). Therefore, T1-MRI imaging of the cardiovascular system, including the heart, aorta, and carotid artery, was possible even 30 minutes after administration to mice. Iron oxide nanoparticles coated with C-DNP-10 (hydrodynamic size: 5 nm) with a surface charge of -30 mV had a hydrodynamic size of 11 nm. Because the nanoparticles had a hydrodynamic size of 8 nm or greater and a surface charge of -30 mV, they accumulated in the liver after administration to mice through phagocytosis by Kupffer cells (macrophages), making them advantageous for liver imaging. This was confirmed using T2-MRI imaging (Figure 41(c)).

[0405] Example 7 In this example, various polysaccharides including Dextran (FIG. 42) were crosslinked to form polysaccharide crosslinked colloidal particles, and a study was carried out to confirm whether these particles could be used as T1 contrast agents.

[0406] 7-1. Nanostructure formation using dextran T-5 180 μmol of dextran T-5 (Figure 42(ai), average molecular weight 5,000 Da) was dissolved in 9 mL of distilled water, and epichlorohydrin and NaOH were added. Diethylenetriamine was then added, and the mixture was stirred at room temperature (RT) for 24 hours, after which it was purified using a 5 kD molecular weight cutoff (MWCO) filter.

[0407] 7-2. Synthesis of nanostructures using maltodextrin All experiments were carried out identically to Example 7-1, except that maltodextrin (Figure 42(a-ii), average molecular weight 990 Da) was used instead of dextran T-5.

[0408] 7-3. Synthesis of nanostructures using alpha-cyclodextrin All experiments were carried out identically except that alpha cyclodextrin (Figure 42(bi), average molecular weight 970 Da) was used instead of dextran T-5 in Example 7-1.

[0409] 7-4. Synthesis of nanostructures using beta-cyclodextrin All experiments were carried out identically, except that beta-cyclodextrin (Figure 42(b-ii), average molecular weight 1100 Da) was used instead of dextran T-5 in Example 7-1.

[0410] 7-5. Synthesis of nanostructures using inulin All experiments were carried out identically except that inulin (FIG. 42(c), average molecular weight 2500 Da) was used instead of dextran T-5 in Example 7-1.

[0411] To confirm the crosslinking of the materials in Examples 7-1 to 7-5, they were prepared at the same concentrations and placed in cuvettes. The absorbance of the materials was measured using a UV-Vis spectrophotometer at wavelengths of 200 to 400 nm. While typical polymers do not absorb light in the visible light range, when nanoparticles are formed after crosslinking, the transmitted light is scattered, resulting in absorption at short wavelengths. As shown in Figure 43, the polymer before crosslinking did not exhibit any absorption in the observed range (orange in Figure 43), but the material with nanostructures exhibited absorption in the short wavelength range (200 to 250 nm), confirming the formation of nanostructures through crosslinking (black in Figure 43).

[0412] The nanostructures synthesized in Examples 7-1 to 7-5 had amine groups formed on their surfaces by diethylenetriamine. To quantify the number of amine groups on the surface per nanostructure, an o-phthalaldehyde assay was performed. The results were 12.7 for dextran T-5, 4.5 for maltodextrin, 8.1 for alpha-cyclodextrin, 9.0 for beta-cyclodextrin, and 8.0 for inulin. Therefore, it was confirmed that amine groups were introduced onto the surface of all nanostructures using this synthesis method.

[0413] 7-6: Introduction of carboxyl groups into nanostructures 25 mg of succinic anhydride was added to the substances of Examples 7-1 to 7-5 at room temperature, and after succinylation reaction for 24 hours, the mixture was purified using a 5 kD molecular weight cutoff (MWCO) filter.

[0414] The number of residual amine groups in the materials of Examples 7-6 was determined by ortho-phthalaldehyde assay. The number of residual amine groups per nanostructure was determined to be 0.7 for dextran T-5, 0.2 for maltodextrin, 0.2 for alpha-cyclodextrin, 0.2 for beta-cyclodextrin, and 0.4 for inulin. A residual amine value of 1 or less means that all amine groups have been replaced with carboxyl groups. Therefore, in the corresponding reactions, all amine groups in nanostructures using various polymers were replaced with carboxyl groups.

[0415] 7-7. Iron incorporation into nanostructures 45 μL of iron chloride hexahydrate solution was added to the material of Example 7-6. The pH was adjusted to 8 using 2.5 M NaOH, and the mixture was reacted at room temperature for 1 hour. After that, the mixture was purified using a 5 kD molecular weight cutoff (MWCO) filter to synthesize nanostructures.

[0416] The iron content and polymer content of the materials in Example 7-7 were analyzed. Surface-bound iron was analyzed using inductively coupled plasma (ICP), and the content of each polymer was analyzed using the phenol-sulfuric acid method. The amounts of iron bound to 1 mg of polymer were analyzed as follows: dextran T-5: 0.03 mg, maltodextrin: 0.026 mg, alpha-cyclodextrin: 0.033 mg, beta-cyclodextrin: 0.026 mg, and inulin: 0.029 mg. This reaction confirmed that similar amounts of iron were introduced during the iron incorporation reaction using various polymers.

[0417] 7-8. Hydration diameter and surface charge measurements of iron-incorporated nanostructures The hydrodynamic diameters of the materials in Example 7-7 were analyzed using dynamic light scattering. Results indicated similar hydration diameters: dextran T-5 (3.6 nm), maltodextrin (6.8 nm), alpha-cyclodextrin (2.9 nm), beta-cyclodextrin (2.8 nm), and inulin (3.8 nm). Surface charge profiles were determined to be -3.01 mV for dextran T-5, -6.62 mV for maltodextrin, -9.06 mV for alpha-cyclodextrin, -7.32 mV for beta-cyclodextrin, and -2.83 mV for inulin. Therefore, the hydration diameters and surface charges of the iron-incorporated nanostructures formed using this synthesis method were similar.

[0418] 7-9. Viscosity measurement comparison of nanostructures The material of Example 7-7 was adjusted to a concentration of 5 mg / mL and then the viscosity was measured at 25° C. The viscosities of the crosslinked polymers were similar: dextran T-5 1.09 mPa s, maltodextrin 1.43 mPa s, alpha cyclodextrin 1.26 mPa s, beta cyclodextrin 1.3 mPa s, and inulin 1.14 mPa s.

[0419] 7-10. Comparison of T1-MRI contrast effects of nanostructures To analyze the T1-MRI performance of the material in Example 7-7, the spin-spin relaxivity coefficient (r2) and spin-lattice relaxivity coefficient (r1) were measured, and their ratio (r2 / r1 ratio) was calculated. The r2 / r1 ratio is a measure of whether a contrast agent is suitable as a T1-MRI contrast agent or a T2 MRI contrast agent. As shown in Figure 44, the materials in Examples 1-10 were analyzed by 3.0 Tesla MRI, and the results were as follows: dextran, r11.90, r22.27, r2 / r1 ratio 1.19; maltodextrin, r14.60, r25.19, r2 / r1 ratio 1.13; alpha-cyclodextrin, r15.26, r25.78, r2 / r1 ratio 1.10; beta-cyclodextrin, r15.62, r26.24, r2 / r1 ratio 1.11; and inulin, r14.33, r24.73, r2 / r1 ratio 1.09. Therefore, all of the synthesized materials had an r2 / r1 ratio close to 1, confirming their T1-MRI contrast effects.

[0420] 7-11. Animal imaging efficacy evaluation of iron-incorporated nanostructures After intravenous administration of the substance of Example 7-7 to mice, T1-weighted images were taken using a 9.4 Tesla MRI. Male Balb / c mice aged 5 weeks or older were anesthetized and the substance of Example 7-7 was administered to the tail vein. T1-MRI images were then taken at various times and the SNR was analyzed. As shown in Figure 45, the iron-incorporated nanostructures were confirmed to exhibit bright signals in the blood vessels (jugular vein) approximately 3 to 5 minutes after injection.

[0421] 7-12. Renal excretion of iron-loaded nanostructures After administering the material of Example 7-7 to animals, T1-weighted images were taken for up to one hour using 9.4 Tesla MRI to confirm renal excretion of the administered material. As shown in Figure 46, most iron-loaded nanostructures showed T1 signals in the bladder within 30 minutes. This indicates that the administered material eventually passes through the kidney and is excreted in the bladder.

Claims

1. A dosage form for non-vascular injection comprising nanoparticles whose hydrated diameter is adjusted to 2 to 20 nm and whose surface charge is adjusted to -20 mV to 0 mV by hydration of hydrophilic groups exposed on the surface so that the nanoparticles do not penetrate or are not discharged into the capillaries at the injection site, and are selectively discharged only into the terminal lymphatic vessels, wherein the nanoparticles are (i) themselves a drug carrier and / or (ii) another drug carrier, and the nanoparticles are not administered intravenously but are discharged only into the lymphatic vessels when injected into a tissue site, thereby providing a drug modality that extends the residence time at the injection site.

2. The non-vascular injectable dosage form according to claim 1, characterized in that the nanoparticles are (a) polysaccharide cross-linked colloidal particles formed by intramolecularly and / or intermolecularly cross-linking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides with a cross-linking agent at the -OH functional groups of the monosaccharide building blocks to prevent hydrolysis by endogenous enzymes, or (b) surface-coated with the polysaccharide cross-linked colloidal particles.

3. The non-vascular injectable dosage form according to claim 1, characterized in that it prolongs the residence time at the injection site, induces an increase in interstitial space at the injection site, an anatomical space expansion, and / or lymph node enlargement.

4. 2. The non-vascular injectable dosage form according to claim 1, wherein the nanoparticles contain at least one component selected from the group consisting of iron, manganese, and gadolinium.

5. The non-vascular injectable dosage form according to claim 1, characterized in that the nanoparticles are designed to be compressible by hydration of the hydrophilic groups exposed on their surface so as to prevent skin reflux or lymph node congestion in the lymphatic vessels at the site of lymphedema.

6. 2. The non-vascular injectable dosage form according to claim 1, wherein the nanoparticles are excreted from the body within one week without remaining at the injection site.

7. 2. The non-vascular injection dosage form according to claim 1, wherein the non-vascular injection method is intradermal, subcutaneous, or direct intralymphatic injection.

8. 2. The non-vascular injectable dosage form according to claim 1, wherein the nanoparticles are excreted from the body by the liver or kidneys.

9. 10. The non-vascularly injectable dosage form of claim 1, which provides a contrast agent for medical imaging procedures used to visualize the lymphatic system and its functions.

10. The non-vascular injectable dosage form according to claim 1, wherein the nanoparticles serve as a contrast agent for diagnosing diseases and effectively imaging biological phenomena at the molecular and cellular levels.

11. 2. The non-vascular injection dosage form according to claim 1, wherein the diameter of the lymphatic vessels through which the nanoparticles are discharged is 0.1 mm or more.

12. 10. The non-vascularly injectable dosage form according to claim 9, characterized in that it is used to assess lymphatic flow, drainage, and congestion.

13. 10. The non-vascularly injectable dosage form according to claim 9, wherein the medical imaging technique is magnetic resonance lymphangiography, ultrasound lymphangiography, computed tomography (CT) lymphangiography, positron emission tomography (PET) lymphangiography, or lymphoscintigraphy.

14. The non-vascular injectable dosage form according to any one of claims 1 to 13, characterized in that the nanoparticles are a contrast agent that exhibits a T1-MRI contrast effect.

15. A non-vascularly injectable contrast agent composition containing nanoparticles whose hydrated diameter is adjusted to 2 to 20 nm and whose surface charge is adjusted to -20 mV to 0 mV so that the nanoparticles do not penetrate or are not discharged into capillaries at the injection site and are selectively discharged only into lymphatic vessels, thereby preventing venous contamination when injected into a tissue site and selectively imaging peripheral to deep body lymphatic vessels.

16. 16. The non-vascularly injectable contrast agent composition according to claim 15, wherein the nanoparticles are (a) polysaccharide-crosslinked colloidal particles formed by intramolecularly and / or intermolecularly crosslinking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides with a crosslinker at the -OH functional groups of the monosaccharide building blocks to prevent hydrolysis by enzymes in the body, and at least one component selected from the group consisting of iron, manganese, and gadolinium is bound to the functional groups derived from the crosslinker exposed on the surface of the polysaccharide-crosslinked colloidal particles; or (b) magnetic nanoparticles coated on the surface with the polysaccharide-crosslinked colloidal particles, and when injected into a tissue site, the polysaccharide-crosslinked colloidal particles are excreted only into the peripheral lymphatics rather than by intravenous administration, thereby extending the residence time at the injection site.

17. 16. The non-vascularly injectable contrast agent composition according to claim 15, wherein the nanoparticles are nano-sized magnetic substances that exhibit T1-MRI contrast effects.

18. 16. The non-vascularly injectable contrast agent composition according to claim 15, characterized in that it is used to visualize lymphatic vessels and / or lymph nodes.

19. The non-vascularly injectable contrast agent composition of claim 15, which provides resolution sufficient to visualize individual lymphatic vessels regardless of depth and the state of subcutaneous tissue.

20. The non-vascularly injectable contrast agent composition according to claim 15, wherein the lymphatic vessels can be observed in T1-MRI images for up to 1 hour after intradermal or subcutaneous injection of the nanoparticles.

21. The non-vascularly injectable contrast agent composition according to claim 15, characterized in that it is used for venous-lymphatic anastomosis.

22. 22. A method for providing information necessary for diagnosing lymphatic diseases without venous contamination, using the non-vascular injection contrast agent composition according to any one of claims 15 to 21.

23. 23. The information providing method according to claim 22, wherein angiography is eliminated before nanoparticles are discharged into veins via lymphatic vessels, thereby enabling angiography noise removal.

24. The information providing method according to claim 22, further comprising providing an image of lymphatic vessel obstruction.

25. 23. The information providing method according to claim 22, wherein information necessary for prevention or early diagnosis of lymphedema is provided.

26. A dosage form for non-vascular injection, characterized in that it contains polysaccharide cross-linked colloidal particles formed by intramolecularly and / or intermolecularly cross-linking 1 to 3 branched polysaccharides or 2 to 30 cyclic polysaccharides with a cross-linking agent at the -OH functional groups of the monosaccharide building blocks, so as to provide a drug modality that is not administered intravenously but is excreted only into the peripheral lymphatics when injected into a tissue site or body cavity, thereby extending the residence time at the injection site after injection to 60 minutes or more. The polysaccharide cross-linked colloidal particles have a hydrated diameter of 2 to 20 nm, preferably 10 nm or less, more preferably 8 nm or less, and even more preferably 6 nm or less, and a surface charge of -20 mV to 0 mV, and are not hydrolyzed by endogenous enzymes.

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