Nanoparticles in which low-molecular-weight heparin and lipid are bound and self-assembled, preparation method therefor, and use thereof

Abstract

The present invention relates to nanoparticles, a preparation method therefor, a pharmaceutical composition containing the nanoparticles, and an anticoagulant containing the composition, the nanoparticles comprising: a low-molecular-weight heparin (LMWH); and a lipid bound to the reducing end of the LMWH, wherein the lipid consists of 4-24 carbon atoms and is self-assembled in an aqueous solution. Specifically, it has been identified that when the nanoparticles are administered in vivo, the anticoagulant effect is higher than that of a conventional LMWH and the effect is sustained over time. Therefore, the nanoparticles, the composition containing the nanoparticles, and the anticoagulant containing the composition can be widely used in various medical fields.

Description

Self-assembled nanoparticles comprising low-molecular-weight heparin and lipid, method for producing the same, and use thereof

[0001] The present invention relates to self-assembled nanoparticles formed by combining low-molecular-weight heparin and lipids, a method for producing the same, and a use thereof. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating thrombotic diseases, which comprises nanoparticles formed by combining low-molecular-weight heparin and lipids and then self-assembling, and which comprises the nanoparticles and has a long-term effect, and an anticoagulant comprising the composition.

[0002] Heparin is a type of sugar, a glycosaminoglycan synthesized in mast cells. It exhibits functions such as preventing blood coagulation and thrombosis by increasing the activity of antithrombin III, a blood factor that inhibits the action of thrombin. Heparin is classified into unfractionated heparin (UFH) and low-molecular weight heparin (LMWH) with a molecular weight of 8000 Da or less, which is produced by fractionating unfractionated heparin.

[0003] Low-molecular-weight heparins are degraded heparins, intended to improve upon the shortcomings of heparin. Their overall half-life is approximately 3 to 5 hours. Methods for improving heparin's half-life include reducing its molecular size and attaching carboxyl groups to it. However, if the molecular size of heparin is reduced to a pentasaccharide or less or its molecular structure is modified, its anticoagulant effect is impaired.

[0004] As a prior art, according to non-patent document 1, very low-molecular-weight heparin (VLMWH) with a half-life of 20 hours has been disclosed, and research on related technologies is continuing, but research on heparin that maintains an anticoagulant effect while improving the half-life is insufficient.

[0005] Against this backdrop, the present invention was completed by confirming that when heparin is bound to a lipid, the half-life is improved while the anticoagulant effect is maintained through the effect of self-assembling in an aqueous solution and moving albumin in the blood.

[0006] [Prior Art Literature]

[0007] [Non-patent literature]

[0008] (Non-patent Document 1) Donat, F. et al. The pharmacokinetics of fondaparinux sodium in healthy volunteers. Clin. Pharmacokinet. 41, 1-9 (2002).

[0009] An object of the present invention is to provide nanoparticles comprising low molecular weight heparin and lipids, which can maintain an anticoagulant effect while improving the half-life.

[0010] Accordingly, an object of the present invention is to provide nanoparticles comprising low-molecular-weight heparin (LMWH); and a lipid bound to the reducing end of the low-molecular-weight heparin, wherein the lipid is a lipid consisting of 4 to 24 carbon atoms, and which self-assemble in an aqueous phase.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating thrombotic diseases comprising the nanoparticles.

[0012] Another object of the present invention is to provide a health functional food composition for preventing or improving thrombotic diseases, comprising the nanoparticles.

[0013] Another object of the present invention is to provide an anticoagulant comprising the pharmaceutical composition.

[0014] Another object of the present invention is to provide a method for producing nanoparticles with improved antithrombotic effects, comprising the steps of (a) forming a compound in which a lipid is bound to the reducing end of low molecular weight heparin; and (b) adding the compound to an aqueous solution to induce self-assembly, wherein the lipid is a lipid having 4 to 24 carbon atoms.

[0015] To achieve the above object, the present invention provides a nanoparticle comprising low-molecular-weight heparin (LMWH); and a lipid bound to the reducing end of the low-molecular-weight heparin, wherein the lipid is a lipid having 4 to 24 carbon atoms, and which self-assembles in an aqueous phase.

[0016] The present invention also provides a pharmaceutical composition for preventing or treating thrombotic diseases comprising the nanoparticles.

[0017] The present invention also provides a health functional food composition for preventing or improving thrombotic disease, comprising the nanoparticles.

[0018] The present invention also provides an anticoagulant comprising the pharmaceutical composition.

[0019] The present invention also provides a method for producing nanoparticles with improved antithrombotic effects, comprising the steps of: (a) forming a compound in which a lipid is bound to the reducing end of low molecular weight heparin; and (b) adding the compound to an aqueous solution to induce self-assembly; wherein the lipid is a lipid having 4 to 24 carbon atoms.

[0020] When the self-assembled nanoparticles and the composition containing the nanoparticles according to the present invention, in which low molecular weight heparin and lipid are combined, are administered to a subject, the anticoagulant effect is maintained and the half-life is improved, and thus, the composition can be widely used as a medicine that can provide a long-term anticoagulant effect in the prevention and treatment of thrombotic diseases.

[0021] Figure 1 is a schematic diagram showing the self-assembly and albumin shuttling effect of LMHO.

[0022] Figure 2 shows the synthesis and characterization of LMWH-lipid conjugates. Figure 2a shows the specific binding of fatty amines to the reducing sugar terminus of LMWH, Figure 2b shows the structure of LMHO according to carbon length, Figures 2c and 2d show the anti-Xa activity of LMWH-lipid conjugates according to carbon length and at various albumin concentrations, and Figures 2e and 2f show the 1D proton NMR results and 2D NMR results of LMWH and LMHO with reducing ends, respectively. 1 H- 13 C HSQC NMR results, Fig. 2g shows the nitrite decomposition for heparin degradation and the insoluble products (lipids) of LMWH and LMHO. 1 Figure 2h shows the results of H NMR, Figure 2h shows the results of mass ratio analysis after nitrite decomposition of LMWH and LMHO, Figure 2i shows the results of non-enzymatic glycosylation comparison, and Figure 2j shows the results of protamine neutralization in buffer or plasma.

[0023] Figure 3 shows the experimental results of the formulation and molecular dynamics analysis of nanoparticles. Figure 3a shows the MD simulation results of self-assembled LMHO with a core hydrophobic octadecylamine moiety in an implicit solvent model, Figure 3b shows the total van der Waals energy and the number of H bonds of LMHO in the MD simulation, Figures 3c to 3f show the particle size distribution, zeta potential, transmission electron microscope image, and FE-SEM image of LMHO nanoparticles, respectively, and Figures 3g and 3h show the fluorescence signal changes and the fluorescence signal changes due to the decomposition of LMHO nanoparticles by DMSO. 1 Figure 3i shows the H NMR results, Figure 3j shows the stability evaluation results of LMHO nanoparticles, and Figure 3j shows the cytotoxicity evaluation results.

[0024] Figure 4 shows the experimental results for the interaction between LMHO and albumin. Figure 4a shows the size distribution of LMHO, Figures 4b and 4c show the results of albumin binding analysis and PAGE analysis, Figure 3d shows the fluorescence intensity of LMWH and LMHO bound to albumin, Figure 3e shows the results of MD simulation for investigating the interaction between LMHO and albumin, and Figures 3f and 3g show transmission electron microscopy images and BLI results between LMHO and albumin.

[0025] Figure 5 shows the in vivo pharmacokinetic analysis and biodistribution of LMHO or LMWH through mouse administration. Figure 5a shows the anti-Xa activity, Figure 5b shows the AUC for the anti-Xa activity, Figure 5c shows the plasma fluorescence intensity after LMWH-RITC or LMHO-RITC administration, Figures 5d and 5e show the in vivo albumin binding analysis and quantitative analysis results through PAGE analysis 1 hour after administration of LMWH or LMHO labeled with Cy 5.5, respectively, Figure 5f shows the results of comparison of clotting times in the tail, Figure 5g shows the results of sustained anticoagulation effect, Figures 5h and 5i show the in vivo distribution and integrated density of LMWO labeled with Cy 5.5, and Figure 5j shows the results of organ-specific fluorescence analysis after administration of LMWO labeled with Cy 5.5.

[0026] Figure 6 shows the results of the systemic and histopathological toxicity evaluation of LMHO. Figure 6a shows the results of comparing the in vivo FXa activity of LMHO neutralized with protamine ( * p < 0.05, ** p < 0.01, *** p < 0.001), Figures 6b and 6c show the liver function parameters including ALT, AST, and Alb in serum and the liver function parameters including A / G, respectively, and Figures 6d and 6e show the H&E staining results (scale bars, 50 μm) and TUNEL staining results (scale bars, 100 μm) by organ.

[0027] Figures 7a and 7b show the structure of LMWH and the structure of a compound synthesized with a fatty amine, respectively.

[0028] Figures 8a and 8b show the 1D of LMWH-lipid compounds after conjugation to the reducing sugar terminal of fatty amine and LMWH, respectively. 1 This shows the H-NMR results.

[0029] Figure 9 shows the UV-vis spectral analysis results of LMWH, octadecylamine, and LMHO.

[0030] Figure 10 shows the number of hydrophobic interaction bonds calculated from molecular dynamic (MD) simulations for self-assembled LMHO nanoparticles.

[0031] Figure 11 shows the results of evaluating the stability of LMHO nanoparticles in DMEM and distilled water.

[0032] Figure 12 shows the results of evaluating the stability of LMWH-lipid compound nanoparticles in serum.

[0033] Figures 13a and 13b show the results of cytotoxicity evaluation of LMWH or LMHO in MRC-5 cells and MDCK.

[0034] Figure 14 shows the size distribution of HSA in the aqueous phase as measured by DLS.

[0035] Figure 15 shows the stability and interaction characteristics after sequential binding simulation of HSA and albumin.

[0036] Figure 16 shows the results of TEM imaging of HSA.

[0037] Figure 17 shows the biodistribution of LMWH. Figures 17a and 17b show the in vivo distribution and integrated density at each time point after subcutaneous administration, respectively, and Figure 17c shows the results of fluorescence analysis showing the distribution by organ.

[0038] Figures 18a to 18c show the results of fluorescence intensity measurements for the organ-specific biodistribution of LMWH or LMHO labeled with Cy 5.5 on day 1, 1 hour, and day 14, respectively.

[0039] Figure 19 shows the percentage of serum electrolytes (sodium, potassium, and chloride) as a renal function parameter at EOWHRKQT.

[0040] Figure 20 shows the histological TUNEL staining results after LMWH and LMHO injection.

[0041] Hereinafter, the present invention will be described in detail.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0043] When the present invention says that a component or a step "includes", this does not mean that other components or other steps are excluded, but rather that other components or other steps may be further included, unless specifically stated otherwise.

[0044] The term "administration" in the present invention means a method of causing at least partial localization to a desired site of an object or placing a given substance into an object. Administration may be carried out by any method known in the art.

[0045] In the present invention, the term “prevention” means any act of suppressing symptoms of a specific disease or delaying its progression by administering the composition of the present invention into the body.

[0046] In the present invention, the term "treatment" may include, without limitation, any act that can improve or benefit symptoms caused by a thrombotic disease by using the composition of the present invention.

[0047] The present invention provides nanoparticles comprising low-molecular-weight heparin (LMWH); and a lipid bound to the reducing end of the low-molecular-weight heparin, wherein the lipid is a lipid having 4 to 24 carbon atoms, and which self-assemble in an aqueous phase.

[0048] The above lipid is an amino compound, and may be included without limitation as long as it is a lipid in which the hydrogen of the hydrocarbon is replaced with an amino group, preferably a lipid including a primary amine group, and more preferably a lipid consisting of 6 to 18 carbon atoms including a primary amine group, but is not limited thereto.

[0049] Additionally, the lipid may further include other functional groups, such as, but not limited to, a hydroxyl group, an ester group, and a urethane group.

[0050] The above low-molecular-weight heparin is heparin made into a low-molecular-weight substance by fractionating unfractionated heparin, and is composed only of short polysaccharide chains, has an average molecular weight of less than 8,000 Da, and can be obtained by fractionation or depolymerization of heparin in a polymeric state, but is not limited thereto.

[0051] In the present invention, the molecular weight of the low molecular weight heparin is 2000 Da to 6000 Da or less, preferably 4000 Da to 6000 Da or less, but is not limited thereto.

[0052] The lipid may include hexylamine, octylamine, decylamine, dodecylamine, octadecylamine or a combination thereof, preferably octadecylamine, but is not limited thereto.

[0053] The above albumin is a protein that plays an important role in maintaining osmotic pressure between blood vessels and tissues by allowing body fluid to remain in the blood vessels. It accounts for 50 to 70% of total serum protein and has a half-life of approximately 20 days.

[0054] Heparin in the above nanoparticles has the characteristic of having an increased half-life when administered into the body, so that the antithrombotic effect is maintained for a long time.

[0055] The average diameter of the above nanoparticles is 50 to 500 nm, preferably 50 to 250 nm, but is not limited thereto.

[0056] The above nanoparticles have binding affinity to serum albumin and can circulate in the bloodstream for 1 to 20 days after binding to albumin, preferably 4 to 14 days, but are not limited thereto.

[0057] The in vivo blood clotting time of the above nanoparticles may be 3000 to 8000 seconds, preferably 4000 to 7000 seconds, but is not limited thereto.

[0058] The anti-FXa activity level of the above nanoparticles may be 0.2 to 0.8 IU / mL, preferably 0.2 to 0.4 IU / mL, but is not limited thereto.

[0059] The half-life of the above nanoparticles may be 10 to 70 hours, preferably 30 to 70 hours, but is not limited thereto.

[0060] The above nanoparticles can be neutralized by protamine.

[0061] The above protamine is used to neutralize the activity of heparin after heparin overdose, cardiac surgery using extracorporeal circulation, or surgical procedures using heparin. Protamine is a strong base and binds to heparin, a strong acid, to form a complex that has no anticoagulant effect.

[0062] The above nanoparticles can form micelle, liposome or nanoaggregate structures by self-assembly, but are not limited thereto.

[0063] The present invention also provides a pharmaceutical composition for preventing or treating thrombotic diseases comprising the nanoparticles.

[0064] The above thrombotic disease may be selected from the group consisting of, but is not limited to, thrombosis, hypertension, stroke, cerebral infarction, angina pectoris, myocardial infarction, arteriosclerosis, peripheral arterial occlusion, renal vein occlusion, central retinal vein occlusion, pulmonary thrombosis, deep vein thrombosis, portal vein thrombosis, cerebral venous sinus thrombosis, cerebral arteriosclerosis, heart disease, ischemic heart disease, intracranial hemorrhage, aneurysm, atherothrombosis, nephrosclerosis, and pulmonary embolism.

[0065] Meanwhile, depending on the mode and method of use of the pharmaceutical composition, the nanoparticles may include a pharmaceutically acceptable salt, and the content of the salt may be appropriately adjusted and used according to the selection of those skilled in the art. In addition, the nanoparticles and the pharmaceutically acceptable salt may be included alone in the pharmaceutical composition, or may be included together with other pharmacologically acceptable carriers, excipients, diluents, or auxiliary ingredients.

[0066] Examples of the pharmaceutically acceptable carrier, excipient or diluent include, but are not limited to, one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin and physiological saline, and any conventional carrier, excipient or diluent may be used. In addition, the pharmaceutical composition may further comprise conventional fillers, bulking agents, binders, disintegrants, anticoagulants, lubricants, wetting agents, pH regulators, nutrients, vitamins, electrolytes, alginic acid and its salts, pectic acid and its salts, protective colloids, glycerin, flavorings, emulsifiers, or preservatives.

[0067] In addition, the pharmaceutical composition may be used as a combination therapy applied simultaneously or at different times, including one or more other therapeutic agents known to be effective in treating or preventing thrombotic diseases in addition to the above-mentioned effective ingredient.

[0068] The pharmaceutical composition of the present invention may be administered orally or parenterally in various dosage forms. When formulating the composition, it may be prepared using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

[0069] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing one or more compounds with at least one excipient, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, or gelatin. For example, tablets or sugar tablets can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture.

[0070] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants in some cases, and anticoagulants, flavoring agents, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, packaging agents, pigments, and preservatives may be additionally included.

[0071] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, or suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0072] The composition of the present invention can be administered orally or parenterally, and can be formulated in the form of an injection for intravenous, intraarterial, intratumoral, intraperitoneal, intramuscular, subcutaneous, intradermal, topical, intranasal, intrapulmonary, and intrarectal administration according to a method known in the art, and can be preferably administered via the intravenous, intraarterial, intratumoral, or subcutaneous route, but is not limited thereto.

[0073] In the case of the above injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for the injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. In addition, the injection may in most cases additionally include isotonic agents such as sugars or sodium chloride.

[0074] The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. That is, the total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered as a fractionated treatment protocol in which multiple doses are administered over a long period of time. It is important to consider all of the above factors and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0075] The present invention also provides a health functional food composition for preventing or improving thrombotic disease, comprising the nanoparticles.

[0076] The above food composition includes all forms, including functional foods, nutritional supplements, health foods, food additives, and feed, and is intended for consumption by humans or animals, including livestock. The above type of food composition can be manufactured in various forms using conventional methods known in the art.

[0077] The above type of food composition can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible plant oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.), and the like. In addition, nutritional supplements include, but are not limited to, capsules, tablets, pills, and the like, and the like, and the like, can be manufactured by adding the compound of the chemical formula 1 of the present invention. In addition, the health functional food is not limited thereto, but for example, the nanoparticles of the present invention can be manufactured in the form of tea, juice, and drinks and consumed by liquefying, granulating, encapsulating, and powdering them so that they can be consumed (health drinks).

[0078] The health food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, the health food of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination.

[0079] The present invention also provides an anticoagulant comprising the pharmaceutical composition.

[0080] Anticoagulants are a type of antithrombotic agent, and antithrombotic agents are classified as anticoagulant agents, antiplatelet agents, or thrombolytics.

[0081] The above antithrombotic agent can prevent the formation of blood clots in blood vessels and dissolve formed blood clots, but is not limited thereto.

[0082] Platelets clump together, and fibrin, a coagulation protein in the blood, acts to clump the platelets together, forming a clot. The anticoagulant can inhibit the action of thrombin, one of the coagulation proteins.

[0083] The above antiplatelet agent can play a role in preventing the recurrence of cerebral infarction by inhibiting the function of platelets, which play an important role in the formation of blood clots within blood vessels.

[0084] The above thrombolytic agent is a drug that dissolves blood clots and can be mainly used for acute myocardial infarction, acute pulmonary embolism, artificial heart valve thrombosis, ischemic stroke, etc.

[0085] The present invention also provides a method for producing nanoparticles with improved antithrombotic effects, comprising the steps of (a) forming a compound in which a lipid is bound to the reducing end of low molecular weight heparin; and (b) adding the compound to an aqueous solution to induce self-assembly, wherein the lipid is a lipid having 4 to 24 carbon atoms.

[0086] The lipid may include hexylamine, octylamine, decylamine, dodecylamine, octadecylamine or a combination thereof through the addition of cyanoborohydride, and is preferably octadecylamine, but is not limited thereto.

[0087] In the above step (a), the low molecular weight heparin may be heated at a reducing end site before binding at 30 to 80°C for 2 to 6 days, preferably at 40 to 70°C for 3 to 6 days, but is not limited thereto.

[0088] In the above step (a), after low-molecular-weight heparin is bound to lipid, the imine at the reducing end of the low-molecular-weight heparin can be reduced to a secondary amine.

[0089] In the above step (b), the compound may form a structure of a micelle, liposome, or nanoaggregate by self-assembly, but is not limited thereto.

[0090]

[0091] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0092]

[0093] [Example 1]

[0094] Synthesis of low-molecular-weight heparin-lipid compounds via position-specific reducing ends of LMWH

[0095] Enoxaparin sodium (LMWH), which is prepared by alkaline hydrolysis among low-molecular-weight heparins, has reactive 2-N,6-O-disulfo-D-glucosamine at the reducing end of the chain and was used in the preparation of the compound according to the present invention. The reducing end of low-molecular-weight heparin was reacted through a one-step protocol of heating at 60°C for 4 days to bind to a lipid amine molecule (Fig. 2a). To reduce imines to secondary amines, the reducing end of low-molecular-weight heparin was selectively reacted with primary amines of various fatty amines with different chain lengths, such as hexylamine (C6), octylamine (C8), decylamine (C10), dodecylamine (C12), and octadecylamine (ODA, C18), in the presence of cyanoborohydride (Fig. 7). As a result of the reaction, low-molecular-weight heparin (LMWH)-lipid compounds with various fatty amine lengths, namely LMWH-C6, LMWH-C8, LMWH-C10, LMWH-C12, and LMWH-C18 (LMHO), were synthesized and purified (Figs. 2b and 7). Typical fatty acids suitable for binding to the binding site of albumin have carbon chains of 12 to 18 carbons, and therefore five saturated medium-chain fatty acids and long-chain fatty acids were used for this purpose. The average reaction yield of the low molecular weight heparin-lipid compounds was approximately 61%.

[0096]

[0097] [Example 2]

[0098] Anti-FXa response assay via albumin binding

[0099] To confirm the therapeutic effects derived from the heparin moiety, the anticoagulant activity of synthesized low-molecular-weight heparin-lipid compounds was evaluated using an anti-FXa assay. The following anti-FXa activities were observed for various low-molecular-weight heparin-lipid compounds (Table 1, Fig. 2c).

[0100] LMWH anti-FXa activity (%) LMWH-C6102 ± 1% LMWH-C899 ± 7% LMWH-C1083 ± 8% LMWH-C1290 ± 4% LMWH-C1897 ± 3%

[0101] These results demonstrate that the anti-FXa activity of low-molecular-weight heparin-lipid compounds is well maintained after synthesis and is independent of the length of the conjugated fatty acid. Subsequently, additional anti-FXa assays were performed with human serum albumin (HSA) at concentrations as low as 0.02% or as high as 2% to assess the lipid-based albumin binding capacity of the low-molecular-weight heparin-lipid compounds. After incubation with albumin, the anti-FXa activity of all low-molecular-weight heparin-lipid compounds decreased, which is thought to be due to binding to albumin. Among these, LMWH-C18 (low-molecular-weight heparin conjugated with octadecylamine, LMHO) exhibited anti-FXa activity of 81.7 ± 1.2% in the presence of 0.02% albumin and 56.3 ± 12.4% in the presence of 2% albumin, which were similar to the results of LMWH-C10 and LMWH-C12 (Fig. 2d). Therefore, considering the anti-FXa activity experimental results (97 ± 3% in Fig. 2b) and the albumin-binding effect, LMHO, which is LMWH-C18 among heparin-lipid compounds, was selected for further low-molecular-weight experiments.

[0102]

[0103] [Example 3]

[0104] Characterization of low-molecular-weight heparin-lipid compounds

[0105] Synthetic and purified low molecular weight heparin-lipid compounds were 1D- 1 H 500 MHz nuclear magnetic resonance (NMR) was used to confirm, and the lipid peak of the compound was confirmed in the range of 0.7-1.3 ppm (Fig. 2e and Fig. 8). In particular, in the case of LMHO 1 H- 13 Two-dimensional 600 MHz NMR with C HSQC = (heteronuclear single-quantum coherence) was used to evaluate the reaction with 2-N,6-O-disulfo-D-glucosamine, the reducing end of heparin, which confirmed that the reducing end site of LMHO was eliminated after the lipid compound was prepared (Fig. 2f).

[0106] The octadecylamine molecule reacted with the aldehyde group transiently generated at the terminal site (Figure 7). Therefore, the HSQC spectrum indicates that the reducing end site of low-molecular-weight heparin is removed, revealing a specific terminal conjugation site of LMHO.

[0107] The successful synthesis of LMHO was further confirmed by nitrous acid digestion, which separated the synthesized compound into a soluble degraded LMWH fragment and a lipid material precipitated from the solvent (Fig. 2g). 1D-1H-NMR analysis confirmed that the precipitate was octadecylamine isolated from LMHO (Fig. 2g). Mass spectrometry analysis of the acid digestion and precipitation revealed that, after lyophilization, 20 mg of LMHO was successfully chemically degraded into 1.14 mg of dry precipitate (lipid) and 18.85 mg of water-soluble material (heparin). Consequently, the ratios of LMWH and ODA in LMHO were determined to be 95.3 ± 0.1% (water-soluble; heparin) and 4.7 ± 0.1% (water-insoluble; lipid), respectively (Fig. 2h). This suggests a calculated molar ratio of 1:0.99, which is almost a 1:1 ratio. According to prior art, the theoretical highest binding ratio is 0.85, which is due to having 15% of the 2-O-sulfo-4-enepyranosuronic acid group at the enoxaparin terminal, but it can be seen that it binds at a slightly higher ratio.

[0108]

[0109] [Example 4]

[0110] Assessment of the reducing end state of LMHO

[0111] Normally, the aldehyde at the end of heparin can react with a primary amine (lipid) to form an imine derivative, also known as a Schiff base, but this was hardly formed in the presence of sodium cyanoborohydride (NaCNBH3) (Figs. 7 and 9).

[0112] The reducing end state of LMHO was further evaluated by detecting the aldehyde group and non-enzymatic glycosylation using Benedict's reagent and LMWH. LMWH or LMHO was dissolved in Benedict's reagent at a concentration of 40 mg / mL. The LMWH solution containing enoxaparin sodium, which has a reducing sugar at the terminal, showed a marked color change, whereas the color of the LMHO solution remained constant (Fig. 2i), indicating that the terminal portion of LMHO was inactivated by the octadecylamine bond.

[0113] For comparison, we investigated another LMWH containing a non-reducing end (NRE-LMWH), nadroparin (average molecular weight: 4.3 kDa), using the same method. The NRE-LMWH molecule possesses a characteristic inactivated NRE (2,5-anhydro-D-mannose) group at the reducing end, as it is chemically depolymerized from unfractionated heparin (UFH) using periodate oxidation. Consequently, the color of the NRE-LMWH solution remained unchanged (Fig. 2i), supporting the end-specific conjugation of octadecylamine to the reducing end of LMHO.

[0114]

[0115] [Example 5]

[0116] In vitro heparin neutralization experiment by protamine

[0117] The neutralizing ability of LMWH and LMHO was investigated by measuring their anticoagulant activity when combined with protamine molecules at two concentration ratios (1:1 and 1:5) in PBS buffer or rat plasma. In the absence of protamine, both LMWH and LMHO consistently exhibited significant anticoagulant activity in buffer (100.7 ± 11 and 96.7 ± 2.5%) and plasma (101 ± 3.6 and 98 ± 13.1%). Treatment with protamine at a 1:1 ratio reduced the anticoagulant activity to 9.7 ± 9.9% (LMWH) and 6.7 ± 7.2% (LMHO), demonstrating high reversibility and neutralizing ability (Fig. 2j). However, the neutralizing capacity in the plasma group was slightly reduced compared to the buffer group, with values ​​of 65.3 ± 3.5% (LMWH) and 45.7 ± 6.0% (LMHO). When treated with protamine at a ratio of 1:5 (heparin / protamine), both the plasma and buffer groups exhibited low anticoagulant activity, with values ​​of 1.7 ± 0.6% (LMWH) and 1.5 ± 0.5% (LMHO) (Fig. 2j). Therefore, LMHO, similar to other heparin molecules, exhibits adequate reversibility in the presence of protamine, demonstrating its potential for therapeutic modulation.

[0118]

[0119] [Example 6]

[0120] Molecular dynamics simulations for self-assembled LMHOs

[0121] We hypothesized that heparin binding to lipid groups could impart amphipathic properties and form self-assembled nanoparticles. To verify the nanoformulation of LMHO in silico, molecular dynamics (MD) simulations were performed from 0 to 500 ps using BIOVIA Discovery Studio software. In the Chemistry at Harvard Macromolecular Mechanics (CHARMM) force field, LMHO molecules formed stable self-assembled nanoparticles over time by arranging lipid groups at the center (Fig. 3a). The calculated van der Waals energy changed from -2119.72 to -2676.28 kcal / m when comparing 2 and 500 s, indicating a transition to a more stable state (Fig. 3b), and the number of hydrophobic interactions increased up to 100 s due to the carbon chain (Fig. 10). The number of H-bonds in the simulated LMHO nanoformulation increased from 53 to 220 (Fig. 3b), a 4.2-fold increase, indicating that the molecular interactions between heparin molecules also became more complex.

[0122]

[0123] [Example 7]

[0124] Characterization of self-assembled LMHO nanoparticles

[0125] The self-assembled nanoparticles of LMHO were directly characterized using dynamic light scattering (DLS). The particle size distribution of LMHO was measured to be 127.1 ± 0.7 nm (Fig. 3c), and the zeta potential was -54.29 ± 4.3 mV (Fig. 3d). Particle observations using TEM and scanning electron microscopy (SEM) imaging revealed spherical shapes of self-assembled LMHO nanoparticles with a size of approximately 200 nm (Figs. 3e and 3f), consistent with the DLS data in Fig. 3c. For more detailed nanoparticle analysis, the fluorescent dye cyanine 5.5 (Cy5.5) was conjugated to the carboxyl group of the LMHO molecule. The formation of these self-assembled nanoparticles was then evaluated by measuring the fluorescence intensity of Cy5.5-conjugated LMHO. Initially, the fluorescence intensity was weak due to self-quenching, and when exposed to DMSO solutions ranging from 0% to 75%, the fluorescence intensity of LMHO increased approximately 23-fold from 294.3 ± 25.1 (DMSO 0%) to 6880.4 ± 207.8 (DMSO 75%) (Fig. 3g). This result demonstrates that DMSO disrupts the hydrophobic interaction of LMHO nanoparticles, thereby increasing the exposure of Cy5.5 fluorescent molecules. Accordingly, the change in ODA intensity is shown. 1 The formation of nanoparticles was confirmed through H-NMR comparative analysis (Fig. 3h). 1 In H-NMR, it was observed that the fatty octadecylamine group, which was not visible in 100% water, was significantly detected as the organic solvent (DMSO) content increased from 0% to 75%.

[0126] To characterize the nanoparticles in more detail, we compared the particle stability of LMHO by measuring particle size at various time points (1, 3, 6, 12, and 24 h). LMHO particles maintained a particle size of less than 200 nm for up to 24 h, demonstrating high particle stability in serum, Dulbecco's Phosphate-Buffered Saline (DPBS), or saline (Fig. 3i). LMHO particles also showed similar results in other solutions, such as DMEM or distilled water (DW), for up to 24 h (Fig. 11). Additionally, we analyzed whether this stability could be due to the long carbon chain in other heparin-lipid compounds. Our measurements revealed that low-molecular-weight heparin-lipid compounds with various lipid lengths (LMWH-C6, -C8, -C10, -C12) exhibited different degrees of particle stability in serum, with no specific pattern observed for carbon chain length (Fig. 12). While LMWH-C6 and LMWH-C12 remained stable, LMWH-C8 and LMWH-C10 increased significantly in size and became unstable after 3 hours (Fig. 12).

[0127]

[0128] [Example 8]

[0129] In vitro toxicity assessment

[0130] To evaluate the cytotoxicity of the synthesized LMHO, cell viability assays were performed on human lung cell lines (MRC-5), canine kidney cell lines (MDCK), and adult human skin fibroblast (HDFa) cell lines, which are fibroblasts isolated from the lung tissue of 14-week-old male embryos of Caucasian origin. Cell viability was as follows when treated with the highest concentration of 100 μg / mL LMWH and LMHO, demonstrating the low cytotoxicity of LMHO (Table 2, Fig. 3j, Fig. 13).

[0131] Cell line LMWHLMHOHDFa cell line 96.0 ± 8.2% 93.6 ± 6.4% MRC-5 cell line 97.4 ± 3.5% 93.2 ± 5.7% MDCK cell line 97.2 ± 5.3% 96.6 ± 6.8%

[0132]

[0133] [Example 9] Evaluation of albumin binding through particle size analysis

[0134] The interaction between LMHO and albumin was confirmed using dynamic light scattering (DLS) by comparing the particle size distributions of albumin at various LMHO-to-albumin ratios of 1:0, 1:1, 1:10, and 1:100. When albumin at a concentration of 0.1 mg / mL was dissolved in DW, the particle size distribution was approximately 6.4 ± 1.1 nm (Fig. 14). However, when LMHO solution was added to this solution at a 1:1 (LMHO / albumin) ratio, the z-average size increased to 9.7 ± 1.5 nm, resulting in nanoparticles (average 100 nm) (Fig. 4a). This could be due to the molecular movement of LMHO, which binds to albumin through hydrophobic interactions, leading to an increase in size. As the percentage of albumin increased, the percentage of 100 nm LMHO particles decreased, indicating that the binding of LMHO increased as the availability of albumin increased (Fig. 4a).

[0135]

[0136] [Example 10]

[0137] Albumin binding affinity assessment

[0138] To compare the binding affinity to albumin, LMWH and LMHO molecules labeled with rhodamine B isothiocyanate (RITC) were evaluated using albumin-coated agarose spin columns. LMWH and LMHO molecules were incubated on the albumin-coated columns for 1 h and then washed four times with DPBS. As a result, the column treated with LMHO-RITC showed a higher intensity than the column treated with LMWH-RITC, indicating that LMHO has a higher affinity for albumin than LMWH (Fig. 4b). The fluorescence intensity of LMWH-RITC was 411.3 ± 10.6 (au), and that of LMHO-RITC was 871.7 ± 46.3 (au), which was two times higher (Fig. 4b).

[0139] The albumin-binding specificity of LMHO was further assessed by measuring the intensity of Cy5.5 bound to albumin in native PAGE after dissolving LMWH-Cy5.5 or LMHO-Cy5.5 with HSA. LMHO-Cy5.5 bound to albumin and loaded slowly, exhibiting high intensity at the top of the gel. In contrast, LMWH-Cy5.5, which did not bind strongly to albumin, was observed at the bottom of the gel, as shown in Figure 4c. This difference demonstrates the higher albumin-binding affinity of LMHO compared to LMWH. LMHO-Cy5.5 exhibited a 3.7-fold increase in the intensity of bound albumin compared to LMWH-Cy5.5 (Figure 4d).

[0140]

[0141] [Example 11]

[0142] Evaluation of the interaction between albumin and LMHO

[0143] To analyze the interaction between albumin and LMHO nanoparticles at the molecular level, we performed MD simulations using albumin and LMHO molecules. First, based on the albumin protein (PDB: 1E7H), docking simulations between LMHO fragments and albumin molecules were performed considering approximately seven lipid-binding sites in albumin. As a result, the LMHO fragments showed a slightly higher binding affinity to the albumin structure (Fig. 15). Next, MD simulations were performed to investigate the interaction between LMHO nanoparticle molecules and albumin protein, and the molecular mechanisms for the formation, movement, and binding of LMHO molecules to albumin were elucidated using BIOVIA Discovery Studio software (Fig. 4e). As a result, the presence of a lipid group sustained the interaction between the compound and albumin, ultimately resulting in binding to the fatty acid-binding site of albumin in a distance-dependent dielectric implicit solvent model.

[0144] The interaction between LMHO and albumin was further confirmed by TEM and bio-layer interferometry (BLI). TEM images showed LMHO nanoparticles with an average size of 214.3 ± 63.1 nm (Fig. 3e), and free albumin was not clearly observed (Fig. 16). Interestingly, after mixing LMHO nanoparticles with albumin, more complex morphologies of nanoparticles and albumin molecules were observed. A large number of nanoparticles bound to or dissociated from albumin were observed, which may explain the dissociation of self-assembled nanoparticles and their transfer to albumin molecules (Fig. 4f). In addition, the interaction between nanoparticles and albumin was analyzed by immobilizing human serum albumin (HAS) on an amine-responsive sensor and using bio-layer interferometry (BLI). Because LMWH does not react with albumin, the KD(M) value of LMWH was not determined in the BLI assay using the HSA molecule. However, LMHO has a KD of 7.53 X 10 -9 It showed a high affinity for albumin molecules, showing a remarkable KD value (Fig. 4g).

[0145]

[0146] [Example 12]

[0147] Pharmacokinetic studies

[0148] The in vivo pharmacokinetics of LMHO and LMWH were evaluated by comparing their anti-FXa profiles. When administered at a dose of 5 mg / kg via the intravenous (IV) or subcutaneous (SC) route, there was no difference in the anti-FXa activity profiles during the first hour after injection. However, LMWH was almost eliminated 8 hours after administration, whereas LMHO remained at approximately 0.23 ± 0.07 IU / mL (IV) and 0.25 ± 0.07 IU / mL (SC) for up to 72 hours (Fig. 5a and Table 3). The half-life (t) of LMHO 1 / 2) was 65.3 hours, which was 45.4 times longer than the half-life of LMWH, which was 1.44 hours. In addition, when the area under the curve (AUC) was calculated using noncompartmental analysis, LMWH and LMHO were 29.13 ± 2.98 h·μg·mL, respectively. -1 and 243.42 ± 26.70 h·μg·mL -1 LMHO showed an AUC that was approximately 8.4 times higher than that of LMHO (Fig. 5b).

[0149] Administration route Dosage (mg / kg) AUC 0-inf (h·μg·mL -1 )CL(mL·h -1 )t 1 / 2 (h)LMWH Intravenous injection 533.06 ± 3.84 38.16 ± 4.5 12.15 ± 0.89LMHO Intravenous injection 5334.15 ± 159.48 4.33 ± 1.9 34 1.17 ± 19.15AUC: Area under the curve, CL: Elimination from the body, t 1 / 2 : Half-life, data are presented as mean ± SD.

[0150] To directly analyze plasma concentrations after injection using dye-conjugated LMWH or LMHO, we analyzed the time course of RITC intensity (3, 6, and 12 hours). LMWH-RITC was rapidly eliminated within 6 hours (11.1 ± 11.2 au), whereas LMHO-RITC maintained high intensity for up to 12 hours (79.7 ± 25.3 au) (Fig. 5c). Because this prolonged action may be due to the combined effects of nanoparticle formation and albumin binding, we performed an albumin-binding assay using plasma. The albumin-binding specificity of LMHO was assessed by measuring the intensity of Cy5.5 bound to albumin in plasma 1 hour after LMHO-Cy5.5 administration. Blot analysis confirmed that LMHO was primarily distributed at the albumin site (Fig. 5d). LMHO-Cy5.5 showed a 5.9-fold increase in the intensity of bound albumin compared to LMWH-Cy5.5 (Fig. 5e).

[0151]

[0152] [Example 13]

[0153] In vivo tail bleeding experiment in mice

[0154] The in vivo anticoagulant effects of LMHO and LMWH were compared through a tail bleeding experiment. LMHO was dissolved in 100 μL of saline at a concentration of 5 mg / mL and administered subcutaneously to mice. The prolongation of clotting time induced by heparin was compared 1 hour later. While the first clotting time of the control group (saline) was 327±183 seconds, the first clotting times of the LMWH and LMHO groups were significantly prolonged to 3145±433 seconds (LMWH) and 4450±2074 seconds (LMHO), respectively. This indicates that LMHO administration delayed the blood clotting time more than LMWH (Fig. 5f).

[0155]

[0156] [Example 14]

[0157] Long-term maintenance of anti-FXa activity after repeated administration

[0158] The sustained anti-FXa activity of LMWH, which has a long half-life, was compared for up to 144 hours through repeated administration. Based on the half-life of LMHO, which was determined to be 65.3 hours, additional administrations were given at 60 and 132 hours. Results showed that the anti-FXa activity of LMWH rapidly decreased within 6 or 12 hours after treatment, with levels persisting below 0.2 IU / mL for 144 hours. In contrast, LMHO exhibited consistent anti-FXa activity levels of 0.2 to 0.4 IU / mL, consistent with its prolonged half-life (Fig. 5g). This suggests that LMHO has the potential to provide long-term, safe application, a property previously unachievable with heparin compounds.

[0159]

[0160] [Example 15]

[0161] In vivo biodistribution of LMHO

[0162] The detailed biodistribution of LMHO in the body was investigated by subcutaneously administering LMHO-Cy5.5 (5 mg / kg), and the fluorescence intensity was measured for 14 days using a fluorescence analyzer (FOBI, ​​Celgenetech, Korea). Unlike LMWH-Cy5.5, most of the subcutaneously injected LMHO-Cy5.5 molecules were observed around the same injection site (Figs. 5h and 17a), and the highest fluorescence intensity was observed 3 h after administration (Figs. 5i and 17b). This indicates dissolution and dispersion of self-assembled heparin nanoparticles, and some may have bound to albumin as single molecules at the nanoscale. Compared with LMWH-Cy5.5, LMHO-Cy5.5 in the kidney exhibited higher fluorescence intensity on day 1 after administration, indicating that excretion and elimination occurred primarily in the kidney (Figs. 5j and 17c). However, LMWH-Cy5.5 showed higher intensity than LMHO-Cy5.5 on day 1 in liver tissue (Fig. 18). Considering that metabolism and excretion of low-molecular-weight heparin occur in the liver, these results suggest that the metabolism of LMHO may be altered.

[0163]

[0164] [Example 16]

[0165] In vivo protamine reversibility experiment

[0166] Protamine sulfate, a positively charged peptide, binds to LMWH and is used to neutralize its anticoagulant effect. The in vivo reversibility of protamine-induced anti-FXa activity was investigated to determine whether LMHO could maintain this activity by reacting with blood protamine and act as an antidote. Three hours after subcutaneous administration of LMHO to mice, protamine sulfate (25 mg / kg) was intravenously injected to neutralize the effect of heparin. As a result, after 3 hours, both LMWH and LMHO showed a decrease in anti-FXa activity due to protamine to 0.09 ± 0.06 IU / mL (LMWH) and 0.003 ± 0.006 IU / mL (LMHO) (Fig. 6a). In the absence of protamine injection, values ​​remained at 0.41 ± 0.01 IU / mL (LMWH) and 0.56 ± 0.03 IU / mL (LMHO).

[0167]

[0168] [Example 17]

[0169] Systemic and histological toxicity of LMHO

[0170] After administration of LMHO (5 mg / kg, SC), systemic toxicity related to the liver and kidney, as well as histological toxicity in various tissues, were evaluated and compared with LMWH and the control group. Neither the LMWH nor LMHO treatment groups showed significant differences from the control group in terms of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine levels (Fig. 6b). Although post-injection albumin analysis revealed altered serum albumin ratios or renal toxicity, no toxicity was observed (Fig. 6c).

[0171] Similarly, the levels of sodium (Na), potassium (K), and chloride (Cl) did not show significant differences compared to the control group (Fig. 19). Furthermore, histological examination using hematoxylin and eosin (H&E) staining of the heart, liver, spleen, lungs, and kidneys showed no differences compared to the control group, demonstrating that no significant systemic or local toxicity was observed in LMHO (Fig. 6d). TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining of the injection site muscles also showed no differences compared to the control group, indicating that heparin can be delivered without serious toxicity (Figs. 6e and 20).

[0172] That is, it can be confirmed that the nanoparticles according to the present invention exhibit high affinity for albumin molecules, and the anticoagulant effect is maintained while the half-life is prolonged.

[0173]

[0174] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Low-molecular-weight heparin (LMWH); and Contains a lipid bound to the reducing end of the above low molecular weight heparin, The above lipid is a lipid consisting of 4 to 24 carbon atoms, Nanoparticles that self-assemble in aqueous solutions.

2. In paragraph 1, Nanoparticles of heparin having a molecular weight of 2000 Da to 6000 Da or less.

3. In paragraph 1, The above lipid is a nanoparticle comprising hexylamine, octylamine, decylamine, dodecylamine, octadecylamine or a combination thereof.

4. In paragraph 1, The heparin in the above nanoparticles has an increased half-life, so that the antithrombotic effect is maintained for a long time.

5. In paragraph 1, Nanoparticles having an average diameter of 50 to 500 nm.

6. In paragraph 1, The above nanoparticles have binding affinity to serum albumin and circulate in the bloodstream for 1 to 20 days after binding.

7. In paragraph 1, Nanoparticles having an in vivo blood clotting time of 3000 to 8000 seconds.

8. In paragraph 1, Nanoparticles having an anti-FXa activity level of 0.2 to 0.8 IU / mL.

9. In paragraph 1, Nanoparticles having a half-life of 10 to 70 hours.

10. In the first paragraph, the nanoparticle is a nanoparticle that can be neutralized by protamine.

11. In the first paragraph, the nanoparticle forms a micelle, liposome or nanoaggregate structure through self-assembly.

12. A pharmaceutical composition for preventing or treating thrombotic disease, comprising a nanoparticle according to any one of claims 1 to 11.

13. In paragraph 12, A pharmaceutical composition for preventing or treating a thrombotic disease, wherein the thrombotic disease is selected from the group consisting of thrombosis, hypertension, stroke, cerebral infarction, angina pectoris, myocardial infarction, arteriosclerosis, peripheral arterial occlusion, renal vein occlusion, central retinal vein occlusion, pulmonary thrombosis, deep vein thrombosis, portal vein thrombosis, cerebral venous sinus thrombosis, cerebral arteriosclerosis, heart disease, ischemic heart disease, intracranial hemorrhage, aneurysm, atherothrombosis, nephrosclerosis, and pulmonary embolism.

14. In paragraph 12, The above pharmaceutical composition is a pharmaceutical composition for preventing or treating thrombotic diseases, which is administered orally or in the form of an injection via an intravenous, intraarterial, intratumoral or subcutaneous route.

15. A health functional food composition for preventing or improving thrombotic disease, comprising the nanoparticle of any one of claims 1 to 11.

16. An anticoagulant comprising the pharmaceutical composition of Article 12. 17.(a) a step of forming a compound in which a lipid is bound to the reducing end of low molecular weight heparin; and (b) a step of adding the compound to an aqueous solution to induce self-assembly; The above lipid is a lipid consisting of 4 to 24 carbon atoms. Method for producing nanoparticles with improved antithrombotic effect.

18. In paragraph 17, A method of manufacturing the lipid, wherein the lipid comprises hexylamine, octylamine, decylamine, dodecylamine, octadecylamine or a combination thereof through the addition of cyanoborohydride.

19. In paragraph 17, A method for producing nanoparticles, wherein in step (a), the low molecular weight heparin is heated at a reducing end site at 30 to 80°C for 2 to 6 days before binding.

20. In paragraph 17, A method for producing nanoparticles, wherein in the step (b) above, the compound forms a micelle, liposome or nanoaggregate structure by self-assembly.

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