Drug delivery system comprising liposomes loaded onto monocytes

Abstract

The present invention relates to a drug delivery system comprising liposomes containing aspirin. The drug delivery system of the present invention is loaded onto monocytes so that only cells that cause inflammation can be targeted, and the monocytes onto which the drug delivery system is loaded can be delivered to platelets while circulating throughout the whole body for a long period of time, thereby having an excellent effect of reducing the activation of platelets.

Description

Drug delivery system comprising liposomes loaded onto monocytes

[0001] The present invention relates to a drug delivery system comprising a liposome loaded onto monocytes and a method for producing the same.

[0002] Aspirin has been used for over a century for its anti-inflammatory and antithrombotic effects, with 100 billion tablets consumed worldwide annually. However, aspirin has the disadvantage of requiring daily administration, as it is broken down in liver cells with a half-life of approximately two hours. While delivery of aspirin to inflamed endothelial cells (ECs) has been shown to restore endothelial dysfunction and exert anti-inflammatory effects, a delivery method that can translate into clinical trials has not yet been established. Furthermore, high doses of aspirin are required to exert anti-inflammatory effects, which can lead to bleeding.

[0003] Meanwhile, atherosclerosis is a disease in which fats, cholesterol, and calcium accumulate in the artery walls, forming plaque. Atherosclerosis progresses slowly, with the thin endothelial layer of the artery's inner wall being damaged by factors such as high blood pressure, smoking, high cholesterol levels, and inflammation. LDL cholesterol penetrates through the damaged endothelial cells, becomes oxidized, and immune cells (macrophages) are activated to remove it. Macrophages transform into oxidized foam cells, which accumulate to form the initial form of a plaque. As the plaque grows, the immune response continues, triggering inflammation and thickening of the artery wall, forming a fibrous cap. This further narrows the blood vessel, impeding blood flow. Over time, the plaque can become unstable and rupture, forming a clot within the bloodstream. If this clot completely blocks the artery, it can lead to a heart attack or stroke.

[0004] Aspirin treatment for atherosclerosis has primarily focused on its antiplatelet effects. A recent clinical trial of over 15,000 patients found that increasing the aspirin dose for the treatment of atherosclerosis did not improve outcomes.

[0005] Therefore, there is an urgent need for the development of a safe method for achieving anti-inflammatory effects using low doses of aspirin and a drug delivery system that can stably deliver aspirin into the body.

[0006] [Prior Art Literature]

[0007] [Non-patent literature]

[0008] Kwang Jae Cho, Korean Journal of Otorhinolaryngology-Head and Neck Surgery 2007;50(7): 562-572)

[0009] In order to solve the above problems, the inventors of the present invention have made extensive efforts to produce liposomes containing aspirin and load them onto monocytes induced by inflammation to deliver aspirin to the affected area targeted by the monocytes.

[0010] As a result, the present invention was completed by developing a drug delivery vehicle including a liposome containing aspirin.

[0011] Accordingly, the purpose of the present invention is to provide a drug delivery vehicle comprising a liposome loaded on monocytes, wherein the liposome contains aspirin therein and has a diameter of 10 to 200 nm.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases, which comprises a drug delivery system as an active ingredient.

[0013] Another object of the present invention is to provide a cell therapy agent for the prevention or treatment of at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases, which comprises a cell containing a drug delivery vehicle, wherein the cell is a monocyte.

[0014] Another object of the present invention is to provide a method for producing a drug delivery vehicle, comprising the steps of adding aspirin to a solution containing liposomes having a diameter of 10 to 200 nm; and stirring the solution.

[0015] To achieve the above purpose, the inventors of the present invention prepared liposomes containing aspirin through the following implementation example, loaded them onto monocytes in a living body, and stably delivered them to sites that induce inflammation, including the liver, spleen, and vascular cells.

[0016] Hereinafter, the configuration of the present invention will be described in detail.

[0017] One aspect of the present invention is a drug delivery vehicle comprising a liposome loaded on a monocyte, wherein the liposome contains aspirin therein and has a diameter of 10 to 200 nm, 10 to 150 nm, 10 to 100 nm, 50 to 200 nm, 50 to 150 nm, or 50 to 100 nm.

[0018] In this specification, a liposome is a self-assembled structure composed of a lipid bilayer, and is an amphipathic molecule with both hydrophobic and hydrophilic portions. Liposomes are highly biocompatible, easy to prepare, and can transport both water-soluble and fat-soluble drugs. Therefore, they are actively being researched as drug delivery vehicles with minimal side effects in the body. Liposomes are chemically stable, non-irritating, and non-toxic, and are structurally similar to the skin's lipid membrane. Furthermore, their surface properties can be varied, allowing them to be used in a variety of applications, including cosmetics, pharmaceuticals, adjuvants, and drug delivery. Liposomes have the advantage of surface modification through conjugation to polymers and ligands. However, liposomes have a low drug loading efficiency, so there are problems such as the drug not being sufficiently loaded into the liposome, the loaded drug being released to the outside, and it being difficult to maintain a stable state in the body.

[0019] In the present invention, liposomes can be classified in various ways according to their properties such as surface charge, size, membrane structure, etc., and for example, the surface charge of liposomes can be determined by a combination of various lipid raw materials such as anionic lipids, neutral lipids, and cationic lipids.

[0020] The anionic lipid may be at least one selected from the group consisting of 1,2-Stearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)], 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, 1,2-dioleoylphosphatidic acid, and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, but is not limited thereto.

[0021] The neutral lipids are cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine. It may be at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA), and phosphatidylcholine (PC), but is not limited thereto.

[0022] In the present invention, the cationic lipid is 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP), dimethyldioctadecylammonium bromide (DDA), 3β[N(N',N'" dimethylaminoethane carbamoyl cholesterol (DC-Chol), 1,2-dioleoyl-3-(dimethylammonium)propane (DODAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-Dimyristoleoyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine; 14:1 Ethyl PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine; 16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine; 18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholin (1,2-distearoyl-sn-glycero-3-ethylphosphocholin; 18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine;1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (16:0 Ethyl PC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 Ethyl PC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholin (12:0 Ethyl PC), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, MVL5), 1,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TA), 1,2-dimyristoyl-3-trimethylammonium-propane (1,2-dimyristoyl-3-trimethylammonium-propane;It may be at least one selected from the group consisting of, but is not limited to, 14:0 TAP) and N4-cholesteryl-spermine (N4-Cholesteryl-Spermine; GL67).

[0023] In one embodiment of the present invention, the liposome composition used dipalmitoyl phosphatidylcholine, cholesterol, and 1,2-steroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]. In the present invention, dipalmitoyl phosphatidylcholine, since the phosphoethanolamine moiety exhibits anionic properties, regulates interactions with drugs or cells, and cholesterol increases the structural stability of the liposome and keeps the liposome more stable against changes in the external environment. In addition, 1,2-steroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] is responsible for structural formation, stably forming a bilayer structure, maintaining the stability of the liposome, and controlling drug release.

[0024] In the present invention, liposomes can be produced by "top-down methods" in which large liposomes are formed and then divided into smaller ones, and "bottom-up methods" in which smaller liposomes are assembled using lipid monomers. Preferably, in order to produce liposomes using the bottom-up method, the liposomes of the present invention can be produced by dissolving lipids in an organic solvent, removing the organic solvent, and rehydrating the lipids in an aqueous solution.

[0025] In the present invention, aspirin may be included in a concentration of 10 to 100 ug, 10 to 80 ug, 10 to 60 ug, 10 to 40 ug, or 10 to 20 ug, but is not limited thereto.

[0026] In the present invention, aspirin can be loaded in an amount of 50 to 100 wt%, 50 to 90 wt%, 50 to 80 wt%, 50 to 70 wt%, for example, 61.5%, relative to the total weight of aspirin (100 wt%) loaded during liposome production.

[0027] In one embodiment of the present invention, when aspirin was treated by liposoming at a concentration of 10 to 100 ug, the expression of COX2 (Cyclooxygenase-2), an important enzyme related to inflammation in vascular endothelial cells, was significantly suppressed, and in particular, when 17.5 ug was treated, COX2 showed particularly high expression suppression.

[0028] In the present invention, monocytes may express one or more markers selected from the group consisting of CD11b, CD14, CD16, CD33, CD68, CD115, and CD62L, for example, a CD11b marker, but are not limited thereto.

[0029] In the present invention, monocytes may be loaded with aspirin through clathrin or caveolin, but are not limited thereto.

[0030] In the present invention, the liposome may additionally include at least one selected from the group consisting of a lipid-lowering agent, an antihypertensive agent, and an antioxidant, but is not limited thereto.

[0031] The above lipid-lowering agents are used to lower blood lipid levels, and may include statins, fibrates, or niacin.

[0032] The above antihypertensive drugs are drugs used to lower blood pressure, and may include ACE inhibitors (Angiotensin-converting enzyme inhibitors), calcium channel blockers, or beta blockers.

[0033] The above antioxidants neutralize free radicals, reduce oxidative stress, and prevent cell damage, and vitamin C, vitamin E, coenzyme Q10, polyphenols, etc. can be used.

[0034] Another aspect of the present invention is a preventive or therapeutic pharmaceutical composition comprising a drug delivery system including a liposome loaded on monocytes as an active ingredient, wherein the preventive or therapeutic pharmaceutical composition is for the prevention or treatment of at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases.

[0035] In the present invention, the inflammatory disease may be at least one selected from the group consisting of septic arthritis, hepatitis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, asthma, systemic lupus erythematosus, atopic dermatitis, and vasculitis, but is not limited thereto.

[0036] In the present invention, cardiovascular disease may be at least one selected from the group consisting of atherosclerosis, coronary artery disease (CAD), myocardial infarction (heart attack), stroke, hypertension, heart failure, arrhythmia, peripheral artery disease (PAD), and valvular heart disease, but is not limited thereto.

[0037] In the present invention, the cerebrovascular disease may be at least one selected from the group consisting of ischemic stroke, hemorrhagic stroke, transient ischemic attack (TIA), cerebral aneurysm, cerebral venous sinus thrombosis (CVST), vascular dementia, and Moyamoya disease, but is not limited thereto.

[0038] As used herein, "atherosclerosis" is a disease in which plaque builds up on the walls of arteries due to the accumulation of fat, cholesterol, and other substances. This disease narrows and hardens blood vessels, impeding blood flow and ultimately causing problems with blood supply to the heart, brain, and other vital organs. Atherosclerosis is a major cause of cardiovascular disease and can lead to serious complications such as myocardial infarction (heart attack), stroke, and peripheral artery disease.

[0039] In one embodiment, the drug delivery vehicle may be as described above.

[0040] In the present invention, the drug delivery vehicle may include a pharmaceutically acceptable salt or solvate thereof together with a liposome containing aspirin.

[0041] As used herein, “prevention” may include any act of inhibiting or delaying the onset of a disease by administering a pharmaceutical composition.

[0042] As used herein, “treatment” may include any action that improves or benefits the symptoms of a disease by administering a pharmaceutical composition.

[0043] In the present invention, treatment with drug delivery systems is characterized by caveolin, a potential mediator of handover to inflammatory cells. Caveolin plays a crucial role in the intracellular uptake of external signaling molecules, and its increased expression in inflammatory cells facilitates the processing of inflammatory signals and responses. Increased inflammatory markers have been shown to increase monocyte recruitment to the site of inflammation.

[0044] The pharmaceutical compositions herein may include chemical or biological compounds or substances for use in the diagnosis, treatment or prevention of a disease.

[0045] In the present invention, the pharmaceutical composition may be formulated and used in various forms according to conventional methods. For example, the pharmaceutical composition may be formulated as an oral dosage form such as a powder, granule, tablet, capsule, suspension, emulsion syrup, etc. Alternatively, the pharmaceutical composition may be formulated as a parenteral dosage form using a diluent or excipient such as a lubricant, wetting agent, flavoring agent, emulsifier, suspending agent, preservative, or surfactant. Alternatively, the pharmaceutical composition may be formulated and used in the form of a topical preparation, suppository, or sterile injection solution.

[0046] In the present invention, the pharmaceutical composition may additionally include conventionally added ingredients, such as conventional supplements and carriers, such as antioxidants, stabilizers, solubilizers, and vitamins, in order to achieve the desired effect.

[0047] Another aspect of the present invention is a preventive or therapeutic cell therapy comprising a cell containing a drug delivery vehicle, wherein the cell is a monocyte, and the cell therapy is for the prevention or treatment of at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases.

[0048] In the present invention, the cell therapy agent may be administered via any conventional route as long as it can reach the target tissue. Parenteral administration may include, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, or intradermal administration.

[0049] In the present invention, the cell therapy agent may be formulated in a suitable form with a pharmaceutical carrier commonly used in cell therapy. The term "pharmaceutically acceptable" refers to a composition that is physiologically tolerable and does not typically cause allergic reactions or similar reactions, such as gastrointestinal upset or dizziness, when administered to humans. Pharmaceutically acceptable carriers include, for example, parenteral carriers such as water, suitable oils, saline solutions, aqueous glucose, and glycols, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Other pharmaceutically acceptable carriers may be referred to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0050] In the present invention, the cell therapy agent may be included in a therapeutically effective amount for the treatment of a disease. The therapeutically effective amount refers to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human, as considered by researchers, veterinarians, doctors, or other clinicians, and includes an amount that induces alleviation of symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the cell therapy agent of the present invention will vary depending on the desired effect. Therefore, the optimal content of the cell therapy agent can be easily determined by those skilled in the art and can be adjusted according to various factors, including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the secretion rate of the composition, the treatment period, and concurrently used drugs. It is important to include an amount that can achieve the maximum effect with the minimum amount without side effects, taking all of the above factors into consideration. For example, the cell therapy agent of the present invention may be 1×10 4 cells / kg to 1×10 8 It can be included in cell / kg.

[0051] Another aspect of the present invention is a method for producing a drug delivery vehicle, comprising the steps of adding aspirin to a solution containing liposomes having a diameter of 10 to 200 nm; and stirring the solution.

[0052] Another aspect of the present invention discloses a method for preventing or treating at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases, comprising the step of administering to a subject in need thereof a pharmaceutical composition comprising a drug delivery system including a liposome loaded on monocytes as an active ingredient.

[0053] In the present invention, the pharmaceutical composition containing a drug delivery vehicle as an active ingredient may be as described above.

[0054] In the present invention, the subject may include a living organism having a disease or suspected of having a disease. For example, the subject may include a human being having or suspected of having cardiovascular disease or atherosclerosis.

[0055] In the present invention, administration may involve introducing a given substance into a subject by any suitable method. For example, administration may include intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. Alternatively, administration may include administration via the oral mucosa.

[0056] In the present invention, administration may include administering to an individual as a single dose or administering to an individual as multiple doses.

[0057] In the present invention, administration may include administering to an individual a different amount of the active ingredient depending on the severity of the disease.

[0058] Another aspect of the present invention discloses a method for preventing or treating at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases, comprising administering to a subject in need thereof a cell therapy agent comprising a cell containing a drug delivery system, wherein the cell is a monocyte.

[0059] In the treatment method of the present invention, the cell therapy agent is administered once or several times a day at a dose of 1×10 4 cells / kg to 1×10 8 It is desirable to include the amount of cells / kg.

[0060] In the treatment method of the present invention, the cell therapy agent of the present invention can be administered in a conventional manner via the rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular, or intradermal route.

[0061] In the present invention, the cell containing the drug delivery vehicle may be as described above.

[0062] The present invention relates to a drug delivery system comprising a liposome loaded onto monocytes, wherein the drug delivery system of the present invention is loaded onto monocytes and can target only cells that cause inflammation, and has an excellent effect in that monocytes loaded with the drug delivery system can circulate throughout the body for a long period of time and be delivered to platelets, thereby reducing platelet activation.

[0063] Figure 1 is a diagram showing the process of loading liposomes into monocytes and delivering them to inflammatory cells according to the target location inside.

[0064] Figure 2 is a diagram and graph showing the results of analyzing liposomes (+ / - aspirin) and the loading ratio of aspirin within the liposomes, taken using dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0065] Figure 3 is a diagram showing the process of measuring liposome uptake by monocytes and the results of liposome uptake after liposomes (+ / - aspirin) were injected into the tail vein of a mouse.

[0066] Figure 4 is a graph and diagram showing the results of liposome uptake by monocytes.

[0067] Figure 5 is a diagram and graph showing the results of evaluating the ex vivo delivery of monocytes to vascular endothelial cells (ECs), hepatocytes, and platelets.

[0068] Figure 6 is a diagram showing that liposomes were delivered from monocytes to vascular endothelial cells where inflammation was induced.

[0069] Figure 7 is a diagram and graph showing whether liposomes were successfully delivered from monocytes to vascular endothelial cells where inflammation was induced.

[0070] Figure 8 is a diagram and graph showing the results of photographing the liposome internalization process of vascular endothelial cells induced by inflammation.

[0071] Figure 9 is a diagram and graph showing increased COX-2 expression in vascular endothelial cells induced by inflammation without aspirin treatment.

[0072] Figure 10 is a diagram and graph showing the results of evaluating solubility and drug delivery effect according to aspirin concentration.

[0073] Figure 11 is a diagram showing the inflammatory mediation process of caveolin.

[0074] Figure 12 is a diagram showing the results of measuring caveolin expression in vascular endothelial cells.

[0075] Figure 13 is a diagram showing the results of measuring the expression levels of caveolin and clathrin in vascular endothelial cells.

[0076] Figure 14 is a diagram showing the results of co-culture of liposome-loaded monocytes with vascular endothelial cells in a transwell.

[0077] Figure 15 is a diagram showing the results of co-culture of liposome-loaded monocytes with vascular endothelial cells in a transwell.

[0078] Figure 16 is a diagram showing the process of internalizing liposomes through inflammatory delivery of caveolin and liposomes in vascular endothelial cells.

[0079] Figure 17 is a diagram and graph showing the results of confirming the functional inhibition of actin, clathrin, or caveolin in transwell culture.

[0080] Figure 18 is a diagram and graph showing the results of co-culturing monocytes with inflammatory endothelial cells in a transwell by loading liposomes with fluorescent tags attached thereto.

[0081] Figure 19 is a diagram and graph showing the process and results for creating a mouse ischemic hind limb.

[0082] Figure 20 is a diagram and graph showing the results of evaluating the improvement effect of ischemic hindlimb after aspirin administration.

[0083] Figure 21 is a diagram and graph showing the results of comparing the therapeutic effects of oral aspirin administration, intravenous administration (liposome-loaded monocytes), and no administration groups in a fatty liver model.

[0084] Figure 22 is a diagram and graph showing the results of comparing the therapeutic effects of oral aspirin administration, intravenous administration (liposome-loaded monocytes), and no administration groups in a fatty liver model.

[0085] Figure 23 is a diagram and graph showing the results comparing the antiplatelet effect of blood after oral and intravenous administration of aspirin to mice.

[0086] Figure 24 is a diagram and graph showing the results after injecting liposomes with fluorescent tags into the tail vein of each mouse.

[0087] Figure 25 is a diagram and graph showing the results of evaluating the in vivo effect of aspirin-liposome tail vein injection on platelet activation compared to oral aspirin administration.

[0088] Figure 26 is a diagram and graph showing the results of evaluating the transfer effect from monocytes to platelets.

[0089] Figure 27 is a diagram showing the manufacturing process of a mouse model that induces atherosclerosis in the carotid artery.

[0090] Figure 28 is a diagram showing the results of intravenous administration of gold-tagged liposomes to a mouse model in which atherosclerosis was induced in the carotid artery.

[0091] Figure 29 is a diagram showing the results of targeted delivery of gold liposomes to atherosclerotic arteries.

[0092] Figure 30 is a diagram showing the delivery of gold liposomes to dysfunctional ECs.

[0093] Figure 31 is a diagram and graph showing changes in the expression of inflammatory markers measured by aspirin delivery.

[0094] Figure 32 is a diagram and graph showing changes in the liver, carotid artery, and blood due to aspirin delivery.

[0095] Figure 33 is a diagram showing histological features according to aspirin delivery.

[0096] Figure 34 is a graph showing the distribution of aspirin-liposomes in the liver.

[0097] Figure 35 is an H&E staining result showing features in the spleen, lung, kidney, intestine, and heart after delivery of aspirin-liposomes.

[0098] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided only to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0099]

[0100] Manufacturing Example 1. Manufacturing of liposomes containing aspirin

[0101] Aspirin solution was prepared by dissolving aspirin (A5376, Sigma-Aldrich, St. Louis, MO, USA) in ethanol (5 mg / mL). The liposome composition was used by dissolving dipalmitoyl phosphatidylcholine (850355P, Sigma-Aldrich), cholesterol (C8667, Sigma-Aldrich), and 1,2-steroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (880120P, Sigma-Aldrich) in a molar ratio of 55:40:5 in ethanol (E7023, Sigma-Aldrich) at 72°C. The mixture was then vigorously stirred at 500 revolutions per minute (RPM) at 72°C for 5 minutes. Next, aspirin solution was rapidly injected into the liposome mixture at 4 times the volume using a syringe needle (20 G; Restek, Bellefonte, PA, USA), and aspirin liposomes were prepared under conditions of vigorous stirring at room temperature for 15 minutes (Fig. 1).

[0102] Aspirin liposomes were collected by size control through filtration, dialysis, and centrifugation. During the filtration process, the solution was passed through three filters in sequence: a drain disk (PETEDD9025, Sterlitech, Auburn, WA, USA), a 0.1 μm membrane filter (PCT019030, Sterlitech), and then another drain disk. This process was repeated seven times using an extruder (GOE-1000 mL, Genizer, Irvine, CA, USA) and a peristaltic pump (BT100L, Lead Fluid Technology, Hebei, China) at a flow rate of 25 mL / min. Next, the liposomes were dialyzed for 24 h in phosphate-buffered saline (PBS; LB004-02, Repligen, Seoul, South Korea) using a 12–14 kDa (132678T, Repligen, Waltham, MA, USA) cut-off membrane to remove any remaining aspirin. After centrifugation, the aspirin liposomes were centrifuged at 30,000 g for 1 h and resuspended in PBS or saline (JW Pharmaceutical, Seoul, South Korea) to collect the aspirin liposomes for experimental use. Aspirin-free liposomes or gold liposomes were prepared by replacing the aspirin solution with PBS or a gold nanoparticle solution (diameter 10 nm; 752584, Sigma-Aldrich). Liposomes were fluorescently tagged with DiD (V22887, Thermo Fisher, Waltham, MA, USA) or DiO (V22886, Thermo Fisher) according to the manufacturer's instructions.

[0103]

[0104] Manufacturing Example 2. Characterization of liposomes containing aspirin

[0105] 2-1. Shape and size distribution of liposomes

[0106] The morphology and size distribution of liposomes containing aspirin were analyzed using transmission electron microscopy (TEM; Jem2100, JEOL, Tokyo, Japan) and dynamic light scattering (DLS; ELS-Z1000, Otsuka Electronics Ltd., Tokyo, Japan).

[0107] Analysis of the liposomes (+ / - aspirin) by dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that they were circular with a diameter of approximately 100 nm (see Fig. 2, a and b).

[0108]

[0109] 2-2. Measurement of the amount of liposomes and the amount of aspirin in the liposomes

[0110] The amount of prepared liposomes was measured using the Stewart assay38. The prepared liposomes and standard lipid samples were dissolved in chloroform (6955, Duksan, Ansan, Republic of Korea), and each sample was reacted with a ferrocyanate reagent prepared by dissolving iron(III) chloride hexahydrate (27.03 g; F2877, Sigma-Aldrich) and ammonium thiocyanate (30.4 g; 221988, Sigma-Aldrich) in deionized water (1 L). The reacted solution was then vigorously stirred for 20 s and centrifuged at 300 g for 10 min to separate the phases. The absorbance (485 nm) of the lower layer was measured using a UV-Vis spectrophotometer (Lambda 25, Perkin Elmer, Waltham, MA, USA). The amount of aspirin in the liposomes was quantified by calculating the area of ​​the aspirin signal intensity period based on the standard curve of aspirin using liquid chromatography-mass spectrometry (LC-MS; Q-exactive orbitrap plus, Thermo Fisher). The loading rate of aspirin loaded inside the liposomes was confirmed to be 61.5% of the total aspirin injected (see Fig. 2, c).

[0111]

[0112] Example 1. Evaluation of monocyte uptake of liposomes containing aspirin

[0113] Liposomes containing aspirin (+ / - aspirin, fluorescent, or gold tag) prepared in Manufacturing Example 1 were injected into the tail vein of each mouse to load the liposomes onto monocytes. Then, 1 day later, monocytes were isolated from the mice, and the distribution of liposomes to monocytes in each organ and spleen was confirmed using an in vivo imaging system (IVIS; 124262, Perkin Elmer). The IVIS confirmed a predominant distribution signal in the liver and spleen (see Fig. 3, c).

[0114] Cells were extracted from the spleen and separated into CD11b-positive (monocyte) and -negative groups. In contrast to the weak signals of other cells, CD11b-positive monocytes exhibited strong fluorescence intensity, indicating liposome uptake (see Fig. 3, d).

[0115] Fluorescence-activated cell sorting (FACS) analysis of monocytes stained with CD11b revealed that 25% of monocytes uptake liposomes within 1 day (see Fig. 4, e). Furthermore, immunostaining with anti-CD11b-FITC (1:200; MA1-10081, Thermo Fisher) revealed the appearance of red (DiD) liposomes between the green membrane and blue nucleus within monocytes (see Fig. 4, f). These results were further confirmed by TEM imaging, which revealed that gold (10 nm)-tagged liposomes within monocytes appear as circular liposomes (see Fig. 4, g).

[0116]

[0117] Example 2. Evaluation of ex vivo delivery of monocytes to vascular endothelial cells (ECs), hepatocytes, and platelets.

[0118] Monocytes (upper chamber) and endothelial cells (EC, lower chamber) were co-cultured for 24 h using Transwells (3422, Corning, New York, NY, USA) with a pore size of 8.0 μm.

[0119] Specifically, endothelial cells were activated to weak and strong activation levels by treating them with lipopolysaccharide (LPS; L4391, Sigma-Aldrich) at concentrations of 0.1 μg / mL and 1 μg / mL, respectively. Delivery into inflamed endothelial cells was confirmed by immunohistochemistry or TEM imaging using gold-tagged liposomes. Aspirin-liposomes (19.2 μg per well in a 24-well plate) were treated with inflamed endothelial cells and compared to treatment with dissolved aspirin (19.2, 38.4, or 96 μg per well in a 24-well plate). Aspirin uptake into inflamed endothelial cells was then measured using LC-MS. Inflammatory endothelial cells were immunostained with anti-cyclooxygenase 2 (COX2) (1:1,000; ab179800, Abcam, Cambridge, MA, USA) antibody and secondary antibody Alexa Fluor 488 (1:500; Jackson Lab, Bar Harbor, ME, USA). Energy-dependent transport was confirmed by immunostaining in transwells at 4°C and 37°C for 6 h. The receptor roles of actin, caveolin, and clathrin in the transport process were verified by treating inflamed endothelial cells with the actin inhibitor cytochalasin D (for actin; 0.1 μM; 8273, Sigma-Aldrich), the caveolin inhibitor nystatin (for caveolin; 50 μg / mL; N6261-500KU, Sigma-Aldrich), or the clathrin inhibitor chlorpromazine hydrochloride (for clathrin; 25 μM; C8138-5G, Sigma-Aldrich). All inhibitors were treated using a 0.1% dimethyl sulfoxide (S-002-M, Merck, Burlington, MA, USA) solution, and monocytes were co-cultured with endothelial cells treated with inhibitors for 30 min for 24 h before immunostaining.High-resolution 3D time-lapse images were acquired with a lattice light-sheet microscope (Lattice Lightsheet 7, Carl-Zeiss, Oberkochen, Germany) and then analyzed using an artificial intelligence (AI)-based automatic segmentation program (Arivis, Carl-Zeiss). Co-localization of liposomes and caveolin was confirmed by staining with anti-caveolin (1:50; arg57976, arigo Biolaboratories Corp, Hsinchu, Taiwan) followed by secondary antibody Alexa Fluor 594 (1:500) using a super-resolution microscope (Elyra 7, Carl-Zeiss). Expression of caveolin and clathrin was evaluated in LPS-treated endothelial cells without co-culture with monocytes by immunostaining with anti-caveolin (1:50), anti-clathrin (1:50; D3C6, Cell Signaling Technology, Danvers, MA, USA), and secondary antibody Alexa Fluor 488 (1:500).

[0120] Hepatocytes were cultured in a transwell system after ethanol (1%) treatment and sealed with parafilm (P7793, Sigma-Aldrich) to establish an inflammatory condition. Cytochalasin D (for actin; 2 μM), nystatin (for caveolin; 50 μg / mL), or chlorpromazine hydrochloride (for clathrin; 50 μg / mL) was used as inhibitors. Platelets were cultured with monocytes in centrifuge tubes (MCT-175-C, Corning) for 3 h, and then subjected to TEM using gold liposomes and immunostaining with anti-CD41-FITC (1:2,000; MA1-80666, Invitrogen), followed by FACS analysis.

[0121] Our results showed that when inflammation was induced by lipopolysaccharide (LPS) or ethanol, inflamed endothelial cells and liver cells (green phalloidin) uptake significantly more liposomes (red) than in non-inflamed cells, as evidenced by co-localization of red liposomes and green cytoskeleton. Furthermore, quantitative image analysis of internalized aspirin-liposomes confirmed their specific delivery to inflammatory cells (Fig. 5). The delivery of liposomes from monocytes to inflammatory endothelial cells was confirmed by TEM images, in which gold (10 nm) dots surround circles within inflammatory endothelial cells (Fig. 6).

[0122] Furthermore, because liposomes may not be internalized but rather adhere only to the membrane surface of inflamed endothelial cells, the liposome delivery process was visualized using 3D high-resolution imaging with a lattice light-seeking microscope. The process of liposomes being delivered from monocytes (blue DiO) and gradually internalized (red DiD) into inflamed endothelial cells (green) over time was shown. Time-series images of a single inflamed endothelial cell revealed that delivery from monocytes was complete by 60 minutes, and another monocyte initiated the process around 70 minutes. Furthermore, automated segmentation using AI-based image analysis confirmed that approximately 70% of all liposomes were internalized through the cell membrane (Figure 7).

[0123] Inflammation-induced vascular endothelial cells were imaged in the same manner several times over 24 hours. As confirmed by quantitative image analysis, a significant increase in liposome internalization was visualized after 6 hours, which was confirmed to persist thereafter (Fig. 8).

[0124] Additionally, the group not treated with aspirin showed a two-fold increase in the expression of cyclooxygenase-2 (COX-2, red) within the inflamed endothelial cells (green actin and blue nucleus). In contrast, the group in which liposomes containing aspirin were delivered to the inflamed endothelial cells via monocytes showed a significant four-fold decrease in the expression of COX-2 (Fig. 9).

[0125]

[0126] Example 3. Evaluation of liposome delivery effect according to aspirin concentration

[0127] The solubility and delivery effects of aspirin were evaluated by adjusting the concentration of aspirin to 19.2 μg, 38.4 μg, and 96 μg (1- to 5-fold: X) in transwell cultures containing inflammatory endothelial cells. The untreated group and liposomes not treated with aspirin were compared as controls.

[0128] Experimental results showed that liposomal delivery (19.2 μg) significantly inhibited COX2 expression in vascular endothelial cells. The same inhibitory effect was observed when the solubilized concentration of aspirin was increased up to 5-fold (96 μg), but significant inhibition began with a 2-fold solubilized treatment. In addition, when the mass of internalized aspirin was measured by liquid chromatography-mass spectrometry (LC-MS), handover enabled cellular uptake of 17.5 μg (91.1%), compared to 3.1 μg (3.2%) with the 5-fold solubilized treatment, demonstrating effective target-specific delivery (Fig. 10).

[0129]

[0130] Example 4. Evaluation of the delivery effect of liposomes containing aspirin according to caveolin expression.

[0131] Liposomes are internalized into cells via i) passive diffusion, ii) actin-mediated invasion (phagocytosis and macrophageization), and iii) receptor-mediated processes (clathrin and caveolin) (Figure 11). Caveolin expression is upregulated in inflammatory conditions, and its expression is significantly increased in inflamed endothelial cells compared to non-inflamed conditions. Therefore, the delivery effect of liposomes containing aspirin was evaluated by assessing the expression of clathrin and caveolin. For the evaluation, liposome-loaded monocytes were cultured with endothelial cells and hepatocytes in a transwell, respectively, and immunostaining was performed using quantitative analysis.

[0132] Our results confirmed that caveolin expression (red) was significantly increased in inflamed endothelial cells compared to non-inflamed cells (see Figures 12 and 13, a). In contrast, clathrin expression was not upregulated by inflammation (see Figure 13, b). In the same environment, inflamed hepatocytes showed a significant increase in caveolin expression compared to the non-inflamed control (see Figure 13, c). As the degree of inflammation increased in the co-culture, more monocytes (yellow boxes: small nuclei) approached endothelial cells (large nuclei), indicating a further increase in the demand for delivery through upregulation of caveolin expression (Figures 14 and 15). Super-resolution microscopy measurements confirmed that caveolin (red) and liposomes (green) colocalized (yellow) in endothelial cells, indicating that caveolin acts as a receptor for liposomes that internalizes liposomes through inflammatory delivery (see Figure 16, d). Furthermore, we confirmed colocalization of gold liposomes and caveolin (Ω-shaped) in vascular endothelial cells by integration following delivery from adjacent monocytes (see Fig. 16, e). In addition, functional inhibition of actin, clathrin, or caveolin was confirmed using cytochalasin D, chlorpromazine, nystatin, and chlorpromazine-nystatin in transwell cultures, respectively (see Fig. 17, f). Addition of red (DiO) fluorescently tagged liposomes significantly reduced liposome uptake by inflamed hepatocytes in all inhibition groups compared to the uninhibited group. Inhibition of actin function resulted in the smallest decrease in liposome uptake among the inhibition groups, and immunostaining results using quantitative analysis showed no significant difference between the receptor-mediated function inhibition groups. In the same environment as EC culture (see Fig. 17, g), similar results were confirmed, except that the synergistic inhibition of clathrin and caveolin was significantly reduced compared to the individual inhibitions.However, as the degree of inflammation increases to a high level, caveolin inhibition significantly reduces uptake compared to clathrin inhibition, confirming the role of caveolin as a receptor. Other comparative results maintained the same trend as in mild inflammation.

[0133] These results demonstrate that caveolin plays a crucial role in the handover process, a role that becomes increasingly important as the severity of inflammation increases. Furthermore, fluorescently tagged liposomes were loaded onto monocytes and co-cultured with inflamed endothelial cells in a transwell. A decrease in the number of liposomes (red DiO) within inflamed endothelial cells was observed when the culture temperature was lowered from 37°C to 4°C (Fig. 18). These results indicate the energy-dependent nature of the handover process as cell energy decreases at 4°C, suggesting that the handover process is mediated by energy-demanding caveolin rather than energy-independent passive diffusion.

[0134]

[0135] Example 5. Mouse Model I for Evaluation of the Aspirin Delivery Process

[0136] Hindlimb ischemia model and fatty liver model were used to evaluate the aspirin delivery process.

[0137] 5-1. Hindlimb ischemia model

[0138] In a hindlimb ischemia model, microchannel hydrogels were implanted as a delivery layer to inflammatory endothelial cells during blood reperfusion. The femoral artery was ligated (day 0). Fluorescently tagged liposomes were injected into the tail vein (day 1). After sacrifice (day 7), liposome targeting by monocyte carriers was examined. Next, the anti-inflammatory effect of aspirin was confirmed through liposome injections (+ / - aspirin) every 3 days (days 2–11) after ligation and sacrifice (day 14) (Fig. 19).

[0139] Specifically, each mouse (C57BL / 6) was implanted with a microchannel hydrogel and femoral artery ligation was performed. The hydrogel was prepared by cross-linking gelatin (5.5% w / v, PBS; G1890, Sigma-Aldrich) and microbial transglutaminase (mTG; 10% w / v, PBS; 1203-50, Modernist Pantry LLC, Elliott, MD, USA) solution (9:1 ratio, final concentration = 5% w / v). As a sacrificial material for mice, poly(N-isopropylacrylamide) (PNIPAM; 535311, Sigma-Aldrich) fibers were placed in a methanol solution (67-56-1, Merck) containing PNIPAM (53% w / v) and a custom-made spinning device (2,500-2,800 RPM) to obtain a density of 11.45 ± 3.13 μg / mm. 3PNIPAM fibers were placed in a mold (4 × 4 × 3 mm) at a density of 10 μm. In this manner, the hydrogel solution was poured into the mold, and threads of PNIPAM fibers were inserted into the gelatin / mTG hydrogel to create a microchannel network. Next, the PNIPAM fibers were dissolved in cold PBS by inducing a gel-to-sol transition, and then perfusion-washed. The hydrogel served as a bed for liposome tracking and handover analysis by enabling the microchannel network to be perfused with surrounding blood vessels. Each C57BL / 6 mouse was anesthetized through a skin incision, and the superior and inferior branches of the femoral artery in the left hind leg were ligated using 6-0 black silk (SK517, Ailey), and the blood vessel between the two branches was then resected. The microchannel hydrogel was implanted in both hind legs, and the skin was sutured using 4-0 black silk (SK434, Ailey). Ischemic hindlimb perfusion was confirmed by laser Doppler imaging (moorLDLS2, Moor Instruments, Devon, UK). Liposomes were injected into the inflamed area via the tail vein, and IVIS tracking was used to evaluate the delivery process to the inflamed area and the anti-inflammatory effect. Microchannel hydrogels were harvested and evaluated by immunohistochemistry using anti-COX2 (1:1,000), anti-tumor necrosis factor-alpha (TNF-α) (1:1,000; 3707, Cell Signaling Technology), anti-interleukin-6 (IL-6) (1:200; NB600-1131, Novus, Centennial CO, USA), and secondary anti-rabbit AlexaFluor 488 (1:500).

[0140] Experimental results confirmed a significant decrease in blood perfusion through the hindlimb, supported by quantitative analysis, using laser Doppler imaging. IVIS imaging showed an increased signal in the hydrogel in the ischemic hindlimb compared to the non-targeted area (see Figure 20, 3b). This result was further confirmed by confocal imaging after harvesting the hydrogel and immunostaining it (see Figure 20, 3c), which showed monocytes (red CD11b) transporting liposomes (green DiD) through the microchannel (dotted line).

[0141] To verify the anti-inflammatory effect, quantitative immunostaining confirmed that COX2 expression was reduced in the ischemic hindlimb 7 days after aspirin administration compared to when no aspirin was administered (see Fig. 20, 3d). This result was confirmed by immunostaining through quantitative analysis that the expression of other inflammatory markers (tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6)) was significantly reduced when aspirin was administered compared to when no aspirin was administered (see Fig. 20, 3e).

[0142] Therefore, the unique properties of monocytes upon loading aspirin-liposomes were confirmed to be effective for efficient targeting and delivery in ischemic hindlimbs.

[0143]

[0144] 5-2. Fatty liver model

[0145] A fatty liver model was developed in apoE KO mice fed a Western diet (D12079B, Research Diets, New Brunswick, NJ, USA) for 42 days. Soluble aspirin and liposomes (+ / - aspirin) were administered orally daily or intravenously weekly via the tail vein, respectively. Livers were harvested and immunostained using anti-TNF-α (1:1,000), anti-IL-6 (1:200), AdipoRed assay reagent (PT-7009, Lonza), anti-reactive oxygen species 581 / 591 (1:200; D3861, Thermo Fisher), and secondary anti-rabbit Alexa Fluor 488 (1:500). Western blotting was performed by treating liver tissues on ice with RIPA buffer (R0278, Sigma-Aldrich) to extract total protein, centrifuging at 13,200 rpm for 30 min, and measuring protein concentration using BCA protein assay (23227, Thermo Fisher). Protein (25 μg) was separated on a 10% SDS-polyacrylamide Mini-PROTEAN TGX gel (456-1084, Bio-Rad Laboratories, Hercules, CA, USA) and electrotransferred to a nitrocellulose membrane using an iBlot 2 NC gel standard stack (IB23001, Invitrogen). Membranes were blocked with 1X triple-buffered saline (TBST) buffer (BTT-9110, T&L, Seoul, Korea) containing 5% nonfat dry skim milk (1706404, Bio-Rad Laboratories) supplemented with 0.1% Tween-20 (P9416, Sigma-Aldrich) for 1 h at room temperature. Membranes were incubated overnight at 4°C with primary mouse anti-COX2 (1:1,000) and mouse anti-actin (1:1,000; sc-47778, Santa Cruz, Dallas, TX, USA) antibodies diluted in 5% nonfat dry skim milk (TBST).Next, the membrane was washed three times (15 min each) with 1X TBST and incubated with secondary goat anti-rabbit IgG (H+L)-orcelain peroxidase (HRP)-conjugated (1:5,000; 31460, Thermo Fisher) or goat anti-mouse IgG (H+L)-HRP-conjugated (1:5,000; 31430, Thermo Fisher) antibody for 1 h. After washing three times with 1X TBST, the blot signals were visualized using Western-enhanced chemiluminescence (ECL) substrate (170-5060, Bio-Rad Laboratories) according to the manufacturer's instructions and analyzed with a luminescent image analyzer (LAS-3,000; 111901, Fuji Film, Tokyo, Japan) and quantified by normalizing the intensity to that of actin.

[0146] As a result of the experiment, compared to the group that was not administered aspirin (see Fig. 21, 3g), the group that was administered only aspirin-liposomes showed a significant decrease in COX2 expression in fatty liver as measured by Western blot analysis. This result was supported by the expression of TNF-α and IL-6 in the same trend as other inflammatory markers in immunostaining analysis (see Fig. 21, 3h and Fig. 22, 4b). In addition, the delivery effect of aspirin-liposomes significantly reduced lipid accumulation and peroxidation as lipotoxicity markers among the test groups (see Fig. 21, 3i and Fig. 22, 4c).

[0147] Through this, it was confirmed that intravenous and oral administration of aspirin significantly reduced IL-6 expression and lipid accumulation compared to cases where aspirin was not administered.

[0148]

[0149] Example 6. Mouse model II for evaluating long-term delivery to platelets

[0150] The antiplatelet effects of oral aspirin, intravenous liposomes, and intravenous aspirin-liposomes in mouse blood were compared (see Figure 23, a). Because the spleen is a major reservoir for monocytes, splenectomy was performed one day before injection to suppress its reservoir function.

[0151] Each mouse was anesthetized, the skin was incised with dissecting scissors, and the major artery and vein of the splenic ligament were ligated with black thread. The distal blood vessels were then severed at the ligation point to remove the entire spleen. Platelets were prepared as described above and activated by treatment with thrombin (372 μM; 10602400001, Sigma-Aldrich) for 10 minutes or incubation on collagen (100 μg / mL; C7521, Sigma-Aldrich)-coated plates for 30 minutes. Platelet analysis was then performed by FACS, scanning electron microscopy (SEM; Carl-Zeiss), IVIS, or immunostaining with anti-PE-CD62p (1:2,000; 12-0626-82, Invitrogen) and anti-FITC-CD41 (1:2,000). As a starting point (see Figure 23, b), DiD- or gold-tagged liposomes were loaded onto monocytes and then incubated with platelets in vitro for 3 h. When DiD-tagged liposomes were detected by FACS (see Figure 23, c), 73.2% of the total platelets (100%) incubated with the liposomes were separated from the monocytes and then uptaken. These results demonstrate transfer from monocytes to platelets.

[0152] After injecting fluorescently tagged liposomes into the tail vein of each mouse, cells were collected from the spleen, the primary site of monocyte distribution, and monocytes (upper tube) were selectively sorted from other cells (lower tube). Fluorescence intensity of each tube was measured in IVIS. Monocytes exhibited a liposome (+) signal representing 75% of the total cells, which persisted for 7 days, demonstrating long-term liposome loading in splenic monocytes (Figure 24). Next, the in vivo inhibition of platelet activation by tail vein injection of aspirin-liposomes was investigated compared to oral aspirin administration. Blood was collected and platelet activation was cultured to determine whether platelets were activated. Splenectomy was then performed to control splenic monocyte depletion, confirming the role of monocytes as carriers in the delivery of circulating platelets. As a result, compared to no aspirin administration, oral aspirin injection and intravenous aspirin-liposome injection showed a significant decrease in platelet activation within a short period (1.5 hours) after injection, as shown in FACS analysis of CD62p expression, a representative marker of platelet activation (see Fig. 25, 4f). After switching from oral aspirin injection to intravenous aspirin-liposome injection, the same trend was observed from day 3 to day 7, and the degree of decrease significantly increased (see Fig. 25, 4g). Splenectomy significantly weakened the antiplatelet effect compared to intact delivery on day 7 due to the absence of the carrier function of splenic monocytes (see Fig. 25, 4h). These results were confirmed by isolating platelets from mice (day 7) and quantitatively analyzing platelet morphology using scanning electron microscopy (SEM) and imaging (see Fig. 25, 4i). Splenectomy revealed the typical morphology of activated platelets, with rough, elongated pseudopodia, unlike intact platelets. These results demonstrate the antiplatelet effect of aspirin-liposomes loaded into splenic monocytes and maintained in circulation for extended periods.After injection of fluorescently tagged liposomes, platelets were isolated from mice and subjected to IVIS analysis (see Figure 25, 4j). The effects of splenectomy were not evident on day 3, but the antiplatelet effect weakened on day 7, indicating a monocyte-dependent long-term therapeutic effect.

[0153] Therefore, transfer from monocytes to platelets is expected to persist for more than 7 days (see Fig. 26, 5b). In contrast, direct targeting effects were also measured by incubating platelets activated after thrombin treatment in vitro with liposomes, aspirin, or aspirin-liposomes for 1 hour (see Fig. 26, 5c). SEM images confirmed that aspirin significantly reduced platelet activation, regardless of liposome encapsulation, compared to liposome treatment alone. These results were confirmed by quantitative image analysis of aeropodia length as a marker of platelet activation spreading. Platelets were activated by incubating them on collagen-coated plates as a model of basal exposure in vascular dysfunction for 1 hour with phosphate-buffered saline (PBS), aspirin, or aspirin-liposomes (see Fig. 26, 5d).

[0154] Quantitative image analysis of platelet aggregation revealed that the expression of a platelet activation marker (green CD41) was significantly reduced in PBS treated with aspirin alone and aspirin-liposomes. These results suggest that aspirin-liposomes can directly target platelets through bloodstream delivery, as the nanoparticles are typically eliminated from the body within 3 days.

[0155]

[0156] Example 7. Targeted and multi-organ therapy with dual action for atherosclerosis

[0157] ApoE KO mice were fed a Western-style diet for 42 days, and partial carotid artery ligation was performed on day 14. Three of the four branches of the left common carotid artery were ligated using sutures (W2814, Ethicon, Raritan, NJ, USA), and the skin was closed with Vicryl sutures (J510G, Ethicon). Fluorescent-tagged liposomes were injected on day 41, regardless of whether splenectomy was performed, and liposome distribution was measured using IVIS. Gold liposomes were injected on day 35, and each organ was removed. Gold concentration and location were measured by inductively coupled plasma mass spectrometry (ICP-MS; Nexion 2000, Perkin Elmer) and transmission electron microscopy (TEM), respectively. Blood was collected from each mouse, and prostaglandin E2 levels were measured using an enzyme-linked immunosorbent assay kit (ab133021, Abcam) according to the manufacturer's instructions. Levels of lipotoxicity or hepatotoxicity markers were measured using an automated clinical chemistry analyzer (FUJI DRI-CHEM NX500i, Fujifilm). Immunostaining was performed with anti-COX2 (1:1,000), anti-TNF-α (1:1,000), anti-IL-6 (1:200), and secondary anti-rabbit Alexa Fluor 488 (1:500) on the collected tissues (see Fig. 27, a). Splenectomy was performed on day 40, and on day 41, fluorescently labeled liposomes were injected, followed by IVIS analysis (see Fig. 27, 5b). In intact mice, liposomes were distributed mainly in the spleen and liver, whereas in splenectomized mice, there was no splenic distribution, with strong and weak signals in the liver and lungs, respectively. Splenectomy resulted in little targeting to the ligated carotid artery in the inflamed state due to depletion of splenic monocytes, as well as relatively weak signal in the unligated artery (Fig. 5c).In contrast, intact mice showed more positive liposome distribution signals in the ligated arteries compared to the unligated control, suggesting that splenic monocytes act as carriers for delivery to the inflamed arteries. In the same model, gold-tagged liposomes were injected on day 35 and analyzed by inductively coupled plasma mass spectrometry (ICP-MS) (days 35, 36, 38, and 42) and transmission electron microscopy (TEM) (day 38) (see Figures 28 and 6a). Results revealed a predominant distribution in the liver and spleen. Compared to the liver, which was set to 100%, the spleen distribution reached 17.11%, confirming efficient liposome targeting to the spleen over a 3-day period compared to other organs. Splenic targeting of gold liposomes increased from day 0 to day 3 (17.11%) and remained at 11.51% until day 7. TEM images (see Fig. 29, 5d) show intracellular gold nanoparticles in monocytes, foam cells (lipid-laden cells derived from monocytes), and endothelial cells, as evidence of targeted delivery to atherosclerotic arteries. TEM images visualize the delivery of gold liposomes to dysfunctional ECs at 3 days post-injection (see Fig. 30, 6b). Because the aortic arch is naturally exposed to flow disturbances, endothelial cells exhibit increased permeability as a sign of dysfunction. Gold liposomes were found within foam cells and endothelial cells, confirming efficient delivery of liposomes to dysfunctional ECs as therapeutic targets. Due to the dual action of the delivery therapy, immunohistochemical staining significantly reduced the expression of inflammatory markers (COX2, TNF-α, and IL-6) in ligated carotid arteries from no aspirin administration to oral aspirin injection and then to aspirin delivery (see Fig. 31, a and Fig. 32, e). There was no significant difference in the expression level of TNF-α between cases where aspirin was not administered and cases where oral aspirin was administered.The same trend was confirmed in the liver with hematoxylin and eosin (H&E) staining, with a significant reduction in lipid droplets following dual-action therapy (see Fig. 32, f). The therapeutic effect of the dual-action was also confirmed in the blood, with significant reductions in total cholesterol, low-density lipoprotein, and triglyceride levels (see Fig. 32, g), as well as in the levels of prostaglandin E2 (PGE2), a blood inflammatory marker (see Fig. 32, h). These multi-organ effects appeared to cooperate to inhibit atherosclerosis in the ligated carotid artery, with H&E analysis showing a significant reduction in lesion formation from no aspirin administration to oral aspirin injection and further to aspirin delivery (see Fig. 32, i). Aspirin delivery was confirmed to produce histological features similar to those of normal (non-inflamed and aspirin-free) inflamed arteries (Fig. 33). Furthermore, the biostability profile of aspirin-liposomes was most predominant in the liver (Fig. 34). Aspirin-liposomes were demonstrated to be non-hepatotoxic, as evidenced by the absence of significant increases in the expression of markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), and alkaline phosphatase (ALP) in blood samples. This result was confirmed by the absence of any abnormal H&E features in the spleen, lungs, kidneys, intestines, and heart (Fig. 35).

Claims

1. A drug delivery vehicle comprising a liposome loaded on monocytes; The above liposome contains aspirin inside, A drug delivery vehicle having a diameter of 10 to 200 nm.

2. In paragraph 1, A drug delivery system wherein the aspirin is included at a concentration of 10 to 100 ug / ml.

3. In paragraph 1, A drug delivery system wherein the above monocytes express at least one marker selected from the group consisting of CD11b, CD14, CD16, CD33, CD68, CD115 and CD62L.

4. In paragraph 1, A drug delivery vehicle, wherein the liposome further comprises at least one selected from the group consisting of a lipid-lowering agent, an antihypertensive agent, and an antioxidant.

5. A pharmaceutical composition for prevention or treatment comprising a drug delivery system according to any one of clauses 1 to 4 as an active ingredient, The above-mentioned pharmaceutical composition for prevention or treatment is a pharmaceutical composition for the prevention or treatment of at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases.

6. A preventive or therapeutic cell therapy product comprising a cell containing a drug delivery system according to any one of claims 1 to 4, The above cells are monocytes, The above cell therapy agent is a cell therapy agent for the prevention or treatment of at least one disease selected from the group consisting of inflammatory diseases, cardiovascular diseases, and cerebrovascular diseases.

7. A step of adding aspirin to a solution containing liposomes having a diameter of 10 to 200 nm according to any one of claims 1 to 4; and A method for producing a drug delivery vehicle, comprising the step of stirring the above solution.

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