Composition for penetrating blood-brain barrier comprising sound-sensitive liposome as active ingredient

Abstract

The present invention relates to a sonosensitive liposome for penetrating a blood-brain barrier, in which the sonosensitive liposome has excellent drug loading efficiency and ultrasound-induced drug release effect, and can effectively penetrate the blood-brain barrier during ultrasound stimulation. In particular, the sonosensitive liposome according to the present invention can be circulated in the body for a long time, thus having a better blood-brain barrier penetration efficiency, and has a high affinity for brain tumor cells, thus having excellent tumor site delivery. Therefore, the sonosensitive liposome according to the present invention can be used as a drug delivery system for delivering a brain disease therapeutic agent or the like to the brain. Specifically, the present inventors have found optimal cavitation conditions capable of stably opening the blood-brain barrier to further enhance the drug delivery effect of the sonosensitive liposome, and thus, if the cavitation conditions are transplanted to the sonosensitive liposome for penetrating the blood-brain barrier according to the present invention, it is expected to obtain an excellent therapeutic effect on various brain diseases.

Description

Technical Field

[0001] This invention relates to compositions comprising acoustic liposomes for penetrating the blood-brain barrier, etc.

[0002] This application claims priority based on Korean Patent Application No. 10-2021-0017524, filed February 8, 2021, and Korean Patent Application No. 10-2022-0015104, filed February 4, 2022. All disclosures in the specifications and drawings of these applications are incorporated herein by reference. Background Technology

[0003] With the increasing incidence of brain tumors, brain infections caused by bacteria or viruses, and neurological disorders, the demand for technologies that can precisely deliver drugs to the brain is also growing. In particular, the most common treatment for brain tumors is currently surgery, which involves opening the skull and removing the tumor.

[0004] However, opening the skull not only puts stress on patients but also raises concerns about the potential for nerve cell damage during surgery. Despite this, surgery remains a viable treatment option because even with brain-targeting drugs, drug delivery is inhibited by the blood-brain barrier (BBB). Therefore, anticancer drugs used to treat brain diseases such as brain tumors are primarily limited to formulations that can cross the BBB, but even these struggle to achieve precise delivery to the disease site, hindering the attainment of complete therapeutic effects. Thus, there is an urgent need to develop technologies that can accurately deliver drugs to brain tissue.

[0005] Recently, global research has been conducted on technologies for delivering drugs to the brain using ultrasound. Cavitation induced by ultrasound and microbubbles (which serve as ultrasound contrast agents) can temporarily open the blood-brain barrier and is being used as a drug delivery method for treating brain diseases. To this end, after injecting microbubbles and then using 3D focused ultrasound, ultrasound is precisely directed to the brain tumor site. This causes the microbubbles located near the blood-brain barrier to locally open it, thereby delivering drugs to brain tissue and triggering the mechanism for treating brain diseases. However, currently used anticancer drugs are small molecule chemotherapeutic agents, which, due to their rapid excretion and absorption by normal tissues after administration, exhibit limitations in their delivery efficacy to the brain.

[0006] Therefore, by developing drug delivery carriers with high blood-brain barrier penetration efficiency and the ability to circulate in the blood for extended periods, and by co-administering them with ultrasound and microbubbles, drugs can be delivered to the brain more effectively. Summary of the Invention

[0007] [Technical Issues]

[0008] As a result of research conducted to address the aforementioned problems, the inventors developed a sonosensitive liposome (IMP302) that can not only effectively cross the blood-brain barrier but also circulate in vivo for extended periods, thereby further enhancing the penetration efficiency of the blood-brain barrier. Furthermore, it exhibits high affinity for brain tumor cells, maximizing the drug delivery effect to the tumor site.

[0009] Therefore, one object of the present invention is to provide a composition for penetrating the blood-brain barrier, comprising acoustic liposomes as an active ingredient.

[0010] Another object of the present invention is to provide a drug delivery carrier comprising acoustic liposomes for penetrating the blood-brain barrier.

[0011] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain diseases, comprising acoustic liposomes.

[0012] Another object of the present invention is to provide a method for manufacturing acoustic liposomes for penetrating the blood-brain barrier.

[0013] However, the technical problems to be solved by the present invention are not limited to the above-mentioned problems, and those skilled in the art will fully understand from the following description other problems not described herein.

[0014] [Technical Solution]

[0015] This invention provides a composition for penetrating the blood-brain barrier, comprising sonosensitive liposomes as the active ingredient. The sonosensitive liposomes comprise DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), DSPE-mPEG2000 (1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), cholesterol, and lyso-PC (lysophosphatidylcholine). The composition comprises a pharmaceutical composition.

[0016] In one embodiment of the present invention, DSPC, DSPE-mPEG2000, DOPE, cholesterol and lyso-PC may be contained in a molar ratio (mol%) of 1-50:1-10:5-80:0.1-50:0.1-20, but are not limited thereto.

[0017] In another embodiment of the invention, the total liposomes may contain 0.1 to 50% dry weight of DSPC.

[0018] It can be based on total liposomes, containing 3 to 50% dry weight of DSPE-mPEG2000.

[0019] It can be based on total liposomes, containing 1 to 80% dry weight of DOPE.

[0020] It can be based on total liposomes, containing 0.05 to 40% cholesterol by dry weight, and

[0021] It can be based on total liposomes, containing 0.5 to 10% dry weight of lyso-PC, but is not limited to this.

[0022] In another embodiment of the invention, the sonosensitive liposome may further comprise at least one selected from the group consisting of sphingolipids and polysorbates, but is not limited thereto.

[0023] Furthermore, the present invention provides a composition for penetrating the blood-brain barrier, comprising sonosensitive liposomes as an active ingredient, the sonosensitive liposomes comprising DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine) and sphingolipids.

[0024] In another embodiment of the invention, the total liposomes may contain DOPE in a molar ratio (%) of 5 to 80, and may contain sphingolipids in a molar ratio (%) of 5 to 80, but are not limited thereto.

[0025] In yet another embodiment of the invention, the sonosensitive liposomes may further comprise DSPE-mPEG2000, but are not limited thereto.

[0026] In yet another embodiment of the invention, DSPE-mPEG2000 may be contained in a molar ratio (%) of 1 to 20 based on total liposomes, but is not limited thereto.

[0027] In yet another embodiment of the invention, the acoustic liposomes may satisfy at least one of the following features selected from the group consisting of, but are not limited thereto:

[0028] (a) Particle size of 100 to 200 nm; and

[0029] (b) The proportion of drug loaded in liposomes to the total added drug is 50% to 100%.

[0030] In another embodiment of the invention, the acoustic liposomes may be able to cross the blood-brain barrier when exposed to ultrasound, but are not limited thereto.

[0031] In another embodiment of the invention, the composition can be used to deliver a drug to the brain, but is not limited thereto.

[0032] In another embodiment of the invention, the drug may be a treatment agent for brain diseases, but is not limited thereto.

[0033] In another embodiment of the invention, the brain disease may be at least one selected from the group consisting of: brain tumors, brain infections caused by bacteria or viruses, Parkinson's disease, encephalitis, stroke, paralysis, Alzheimer's disease, Lujarig's disease, Huntington's disease, Pick's disease, Kreutzfeldt-Jacob's disease, epilepsy, thrombosis, embolism, cerebral infarction, small artery occlusion, and brain metabolic disorders, but is not limited thereto.

[0034] In another embodiment of the invention, the brain disease treatment agent may comprise at least one selected from the group consisting of: vincristine, vinblastine, vinflunine, vindesine, vinorelbine, temozolomide, carmustine, lomustine, cabazitaxel, docetaxel, larotaxel, oxalitaxel, paclitaxel, temozolomide, ixaspirin, lomustine, procarbazine, rituximab, tocilizumab, temozolomide, carboplatin, erlotinib, irinotecan, enzatolin, vorinostatin, doxorubicin, cisplatin, glimepiride, 5 - Fluorouracil, tamoxifen, topotecan, belotecone, imatinib, fluorouracil, gemcitabine, leuprorelin, flutamide, zoledronic acid, methotrexate, camptothecin, hydroxyurea, streptozocin, penoxuridine, retinic acid, nitrogen mustard, chlorambucil, busulfan, doxyfluridine, mitomycin, prednisone, everolimus, mitoxantrone, levodopa, carbidopa, entacapone, tocapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics, and amantadine, but not limited to these.

[0035] In another embodiment of the invention, the sonosensitive liposomes can be hydrated with ammonium sulfate, ammonium citrate or TEA-SOS, but are not limited thereto.

[0036] In another embodiment of the invention, the composition may be applied sequentially or simultaneously with ultrasonic treatment, but is not limited thereto.

[0037] In yet another embodiment of the invention, the ultrasound may satisfy, but is not limited to, at least one of the following features:

[0038] (a) The frequency of the ultrasound is from 20 kHz to 3 MHz; and

[0039] (b) Duty cycle of 0.5% to 20%.

[0040] In another embodiment of the invention, ultrasonic treatment may occur sequentially with or simultaneously with the application of microbubbles, but is not limited thereto.

[0041] Furthermore, the present invention provides a drug delivery carrier for penetrating the blood-brain barrier, comprising acoustic liposomes as the active ingredient. The acoustic liposomes comprise DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), DSPE-mPEG2000 (1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), cholesterol, and lyso-PC (lysophosphatidylcholine).

[0042] Furthermore, the present invention provides a drug delivery carrier for penetrating the blood-brain barrier, comprising sonosensitive liposomes as the active ingredient. The sonosensitive liposomes comprise DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine) and sphingolipids.

[0043] Furthermore, the present invention provides a method for producing a composition for penetrating the blood-brain barrier, comprising the following steps:

[0044] (S1) Dissolve at least one selected from the group consisting of DSPC, DSPE-mPEG2000, DOPE, cholesterol and lyso-PC in a first organic solvent;

[0045] (S2) Evaporate the organic solvent to produce a liposome membrane; and

[0046] (S3) Hydrate the liposome membrane with an aqueous solution.

[0047] In one embodiment of the present invention, the first organic solvent in step (S1) may be at least one selected from the group consisting of: dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol and diethyl ether, but is not limited thereto.

[0048] In another embodiment of the invention, the first organic solvent in step (S1) may be supplemented with polysorbate, but is not limited thereto.

[0049] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of brain diseases, comprising acoustic liposomes as an active ingredient. Preferably, the acoustic liposomes comprise a drug.

[0050] Furthermore, the present invention provides a method for preventing or treating brain diseases, comprising the step of administering an effective dose of acoustic liposomes to a subject in need. Preferably, the method may further include the steps of processing with ultrasound and / or administering microbubbles.

[0051] Furthermore, this invention provides the use of acoustic liposomes for the prevention or treatment of brain diseases.

[0052] Furthermore, the present invention provides the use of the composition in the manufacture of therapeutic agents for brain diseases.

[0053] Furthermore, the present invention provides a method for delivering a drug to the brain, comprising the step of administering an effective dose of a drug-loaded acoustic liposome to a subject in need. Preferably, the method may further include the steps of processing with ultrasound and / or administering microbubbles.

[0054] Furthermore, the present invention provides the use of acoustic liposomes for delivering drugs to the brain.

[0055] Furthermore, this invention provides the use of acoustic liposomes in the manufacture of brain-targeting drug delivery carriers.

[0056] [Beneficial Effects]

[0057] This invention relates to acoustic liposomes for penetrating the blood-brain barrier. Acoustic liposomes not only exhibit excellent drug encapsulation efficiency and drug release under ultrasound stimulation, but also effectively penetrate the blood-brain barrier when stimulated by ultrasound. In particular, the acoustic liposomes according to the invention can maintain long-term circulation in vivo, resulting in excellent blood-brain barrier penetration efficiency, and have a high affinity for brain tumor cells, thereby providing excellent delivery to the tumor site. Therefore, the acoustic liposomes according to the invention can be used as drug delivery carriers for delivering therapeutic agents for brain diseases to the brain. Specifically, the inventors of the invention have determined optimal cavitation conditions that stably open the blood-brain barrier, aiming to further enhance the drug delivery effect of the acoustic liposomes. It is anticipated that by combining these cavitation conditions with the acoustic liposomes according to the invention for penetrating the blood-brain barrier, excellent therapeutic effects can be achieved in various brain diseases. Attached Figure Description

[0058] Figure 1 The results of a quantitative analysis using FACS of the penetration extent of sonosensitive liposomes, Doxil liposomes and Marquibo liposomes according to the present invention for penetrating the blood-brain barrier into brain tumor cells are shown.

[0059] Figure 2a The results of observing the brain tumor cell infiltration effects of acoustic liposomes and Doxil liposomes according to the present invention for penetrating the blood-brain barrier by confocal fluorescence microscopy are shown (red: liposomes, blue: cell nuclei, magnification: 10X).

[0060] Figure 2b Another result is shown, obtained by confocal fluorescence microscopy, of the brain tumor cell infiltration effect of acoustic liposomes and Doxil liposomes according to the present invention for penetrating the blood-brain barrier (red: liposomes, blue: cell nuclei, magnification: 10X).

[0061] Figure 3 The results show the results of MTT analysis confirming the anticancer killing effect of sonic liposomes, Doxil liposomes and free doxorubicin (free DOX) according to the present invention on brain tumor cells subjected to ultrasound treatment.

[0062] Figure 4a Images of brain tissue infiltration resulting from Evans blue (EB) dye penetration based on microbubble number are shown, along with the results of a quantitative analysis of infiltration efficiency.

[0063] Figure 4b The results of H&E staining are shown to verify the stability of brain tissue based on the number of microvesicles.

[0064] Figure 5a Images of brain tissue infiltration caused by Evans blue dye penetration of the blood-brain barrier, based on ultrasound parameters, are shown.

[0065] Figure 5b The results show the results of a quantitative analysis of the infiltration efficiency of Evans blue dye through the blood-brain barrier based on ultrasound parameters.

[0066] Figure 5c The results of H&E staining are shown to verify the stability of brain tissue based on ultrasound parameters.

[0067] Figure 6 Results confirming the brain tissue delivery efficacy and distribution patterns of the acoustic liposomes and Marquibo liposomes for penetrating the blood-brain barrier according to the present invention in mice.

[0068] Figure 7 Results are shown confirming the distribution patterns of liposomes without sphingomyelin (IMP302-004) and liposomes containing sphingomyelin (IMP302-005) in major organs based on ultrasound parameters.

[0069] Figure 8 Results comparing the blood-brain barrier penetration effects of acoustic liposomes (IMP302-004) and Doxil liposomes according to the present invention are shown. Detailed Implementation

[0070] This invention relates to compositions for penetrating the blood-brain barrier, and to the development of acoustic liposomes that not only exhibit excellent efficiency in drug encapsulation and ultrasound-induced drug release, but also effectively penetrate the blood-brain barrier when stimulated by ultrasound.

[0071] Specifically, in one embodiment of the invention, it was demonstrated that in aqueous liposomes (ammonium sulfate, ammonium citrate, and TEA-SOS), all three types of liposomes exhibited a particle size of approximately 200 nm and excellent drug encapsulation efficiency. In particular, loading drugs with an aqueous solution of ammonium sulfate or ammonium citrate at 60°C for 2 hours was shown to be the optimal condition for drug encapsulation in liposomes (Example 1).

[0072] In another embodiment of the invention, a comparison of the physical properties and characteristics of liposomes based on the type of lipids constituting the liposomes revealed that liposomes manufactured using at least one of the following groups exhibited the best drug encapsulation efficiency and sonic responsiveness: (i) DOPE; and (ii) DSPC, DSPE-mPEG2000, cholesterol, lyso-PC, and sphingomyelin (Example 2).

[0073] In another embodiment of the invention, when the acoustic liposomes according to the invention were co-cultured with brain tumor cell lines, it was demonstrated that the liposomes could effectively penetrate into brain tumor cells (Examples 3 and 4).

[0074] In another embodiment of the present invention, when acoustic liposomes containing anticancer drugs were co-cultured with brain tumor cell lines while ultrasound was applied, the acoustic liposomes were shown to exhibit excellent anticancer effects on brain tumor cells (Example 5).

[0075] In another embodiment of the invention, in order to further enhance the drug delivery effect of acoustic liposomes to the brain, parameters of acoustic cavitation and microbubbles that can stably open the blood-brain barrier were determined (Examples 6 and 7).

[0076] In another embodiment of the invention, when the acoustic liposomes according to the invention were administered in a mouse model while the blood-brain barrier was opened by applying ultrasound and microbubbles, it was demonstrated that the liposomes effectively penetrated into the brain tissue (Examples 8 and 9).

[0077] Therefore, the acoustic liposomes for penetrating the blood-brain barrier according to the present invention have been shown to accurately deliver encapsulated drugs to the brain by effectively crossing the blood-brain barrier, and are expected to be used as drug delivery carriers for treating a variety of diseases.

[0078] The present invention will now be described in detail.

[0079] This invention provides compositions and / or drug delivery carriers for penetrating the blood-brain barrier, comprising acoustic liposomes containing at least one selected from the group consisting of: DSPC (1,2-distearyl-sn-glycerol-3-phosphate choline), DSPE-mPEG2000 (1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), cholesterol, lyso-PC (lysophosphatidylcholine), and sphingolipids. The composition comprises a pharmaceutical composition.

[0080] Preferably, the present invention provides:

[0081] i) A composition for penetrating the blood-brain barrier, comprising sonosensitive liposomes as an active ingredient, said sonosensitive liposomes comprising DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), DSPE-mPEG2000 (1,2-distearyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), DOPE (1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine), cholesterol, and lyso-PC (lysophosphatidylcholine);

[0082] ii) A composition for penetrating the blood-brain barrier, comprising sonosensitive liposomes as an active ingredient, said sonosensitive liposomes comprising DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine) and sphingolipids; and / or

[0083] iii) A composition for penetrating the blood-brain barrier, comprising a sonosensitive liposome as an active ingredient, said sonosensitive liposome comprising DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine), sphingolipids and DSPE-mPEG2000.

[0084] In one embodiment of the invention, the sonosensitive liposome may further comprise polysorbate.

[0085] In this invention, "DOPE" is an abbreviation for 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, wherein the lipid is known to form heteroliposomes with DOTAP, which is used as a drug delivery carrier.

[0086] In this invention, "DSPC" is an abbreviation for 1,2-distearate-sn-glycerol-3-phosphocholine, which refers to a phospholipid composed of two stearic acids attached to the head group of phosphatidylcholine.

[0087] In this invention, "DSPE-mPEG2000" is an abbreviation for 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine conjugated with methoxy (polyethylene glycol 2000), referring to a polyethylene glycol derivative of 1,2-distearyl-sn-glycerol-3-PE (DSPE).

[0088] In this invention, "cholesterol" refers to a lipid present in the cell membrane of all animal cells and is one of the sterols (modified steroids). The cholesterol according to this invention includes cholesterol derivatives. Cholesterol derivatives may include, for example, sitosterol, ergosterol, stigmasterol, 4,22-stigmasteroldien-3-one, stigmasterol acetate, lanosterol, cycloartenol, or combinations thereof. Cholesterol is located in the lipid bilayer, its amount can be modulated to reduce or increase permeability, and it can be used independently of its proportion in liposomes.

[0089] In this invention, "Lyso-PC" is an abbreviation for lysophosphatidylcholine, which is a phospholipin derived from phosphocholine with a head group composed of choline. It represents a lipid composed of a monoacyl chain, and there are known to be many types of Lyso-PC.

[0090] In this invention, lyso-PC can be represented by chemical formula 1 or 2, but is not limited thereto. Preferably, lyso-PC can be 1-LPC (lysophosphatidylcholine) or 2-LPC represented by chemical formula 1 or 2, but is not limited thereto.

[0091] [Chemical Formula 1]

[0092]

[0093] [Chemical Formula 2]

[0094]

[0095] (In chemical formula 1 or 2, the R mentioned in this article can be C6 to C6.) 26 Alkyl, C6 to C 26 alkenyl, C6 to C 26 Alkyne group, substituted or unsubstituted C6 to C 26 Cycloalkyl, substituted or unsubstituted C6 to C 26 aryl, substituted or unsubstituted C7 to C 26 Arylalkyl or H.

[0096] In this invention, lyso-PC can be at least one selected from the group consisting of: lyso-PC(6:0), lyso-PC(7:0), lyso-PC(8:0), lyso-PC(9:0), lyso-PC(10:0), lyso-PC(11:0), lyso-PC(12:0), lyso-PC(13:0), lyso-PC(14:0), lyso-PC(15:0), lyso-PC(16:0), 2-lyso-PC(16:0), lyso-PC(17:0), lyso-PC(18:0), lyso-PC(1 ...2:0), lyso-PC(13:0), lyso-PC(14:0), lyso-PC(15:0), lyso-PC(16:0), lyso-PC(17:0), lyso-PC(18:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC(19:0), lyso-PC( 0), lyso-PC(17:1), lyso-PC(18:0), lyso-PC(18:1), lyso-PC(18:2), 2-lyso-PC(18:0), 2-lyso-PC(18:1), lyso-PC(19:0) , lyso-PC(20:1), lyso-PC(20:4), lyso-PC(22:0), lyso-PC(22:5), lyso-PC(24:0), lyso-PC(26:0) and lyso-PC(20:5), but not limited thereto.

[0097] Furthermore, preferably, lyso-PC can be MSPC (1-stearoyl-2-lyso-sn-glycerol-3-phosphocholine), but is not limited thereto.

[0098] In this invention, "sphingolipid" refers to a lipid containing a sphingosine base backbone, to which fatty acids can be attached via amide bonds and head groups of primary hydroxyl groups. Sphingolipids are found in all animals, plants, fungi, and some protozoa or viruses, and are known to play an important role in maintaining the structure and function of cell membranes and in transmitting signals between cells. In this invention, sphingolipids are not limited to any particular type, but are preferably at least one selected from the group consisting of sphingosine, ceramides, sphingomyelin, cerebrosides, and gangliosides.

[0099] Preferably, the sphingolipid according to the invention is a sphingomyelin. In this invention, "sphingomyelin" refers to sphingosine phospholipids composed of phosphocholine and ceramide or phosphoethanolamine head groups. Natural sphingomyelins are found in animal cell membranes, especially in the membrane sheath surrounding nerve cell axons.

[0100] In this invention, "polysorbate" refers to a nonionic surfactant derived from ethoxylated dehydrated sorbitol esterified with fatty acids. Polysorbates are well known to act as emulsifying agents and solubilizers in liposomes. In the case of sonosensitive liposomes according to the invention, the efficiency of blood-brain barrier penetration can be improved by further incorporating polysorbates. In this invention, the polysorbate may be selected from, but is not limited to, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. Preferably, the polysorbate mentioned herein is polysorbate 80.

[0101] In one embodiment of the present invention, DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC may be contained in molar ratios (mol%) of 1 to 50, 1 to 10, 5 to 80, 0.1 to 50, and 0.1 to 20, but are not limited thereto. The inventors have demonstrated through specific experiments that liposomes meeting these compositional ratios exhibit excellent drug encapsulation efficiency, ultrasound responsiveness, and blood-brain barrier permeability.

[0102] More specifically, the molar ratios of DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC are 1–50:1–10:5–80:0.1–50:0.1–20 mol%, 1–50:1–10:5–68:0.1–50:0.1–20 mol%, 1–50:10–15:5–80:0.1–50:0.1–20 mol%, and 1–50:1–20:5–70:0.1– 35:0.1~15 mol%, 1~50:1~10:5~70:0.1~50:0.1~20 mol%, 1~40:1~9:35~80:0.1~40:1~10 mol%, 1~40:1~10:20~70:0.1~50:1~10 mol%, 1~40:1~10:20~68:0.1~50:1~8 mol%, 1~30:1~10:10~80:0.1~50:0. 1–20 mol%, 1–30: 1–9: 5–70: 0.1–40: 0.1–20 mol%, 1–30: 1–9: 5–68: 0.1–40: 0.1–20 mol%, 1–30: 1–10: 20–80: 0.1–50: 2–10 mol%, 1–30: 1–20: 20–68: 0.1–40: 1–11 mol%, 1–20: 1–10: 15–80: 1–48: 1–12 mol%. 10~40:3~7:40~68:0.1~45:0.1~11 mol%, 0.1~20:1~8:63~67:0.1~32:3~11 mol%, 5~15:1~7:60~67:10~32:1~11 mol%, 8~12:3~6:50~66:10~32:5~11 mol%, or 0.1~20:1~8:53~57:0.1~30:5~10 mol%, but not limited to these.

[0103] Additionally, in this invention, based on total liposomes, the liposomes may contain 1 to 50 mol%, 1 to 40 mol%, 1 to 30 mol%, 1 to 25 mol%, 1 to 20 mol%, 1 to 15 mol%, 1 to 7 mol%, 5 to 50 mol%, 5 to 40 mol%, 5 to 35 mol%, 5 to 30 mol%, 5 to 25 mol%, 5 to 20 mol%, 5 to 15 mol%, 7 to 12 mol%, 25 to 35 mol%, 27 to 32 mol%, or 35 to 45 mol% of DSPC, but are not limited thereto.

[0104] In addition, in this invention, based on total liposomes, DSPE-mPEG2000 may be contained in 1 to 10 mol%, 1 to 9 mol%, 1 to 8 mol%, 1 to 7 mol%, 1 to 6 mol%, 1 to 5 mol%, 2 to 9 mol%, 2 to 8 mol%, 2 to 7 mol%, 2 to 6 mol%, 2 to 5 mol%, 3 to 8 mol%, 3 to 7 mol%, or 4 to 6 mol%, but is not limited thereto.

[0105] Furthermore, in this invention, based on total liposomes, the liposomes may contain 5 to 80 mol%, 5 to 75 mol%, 5 to 70 mol%, 5 to 68 mol%, 10 to 75 mol%, 10 to 70 mol%, 10 to 68 mol%, 20 to 75 mol%, 20 to 70 mol%, 20 to 68 mol%, 30 to 75 mol%, 30 to 70 mol%, 30 to 68 mol%, 40 to 80 mol%, 40 to 75 mol%, 40 to 70 mol%, 40 to 68 mol%, 45 to 80 mol%, 45 to 70 mol%, 50 to 80 mol%, 50 to 70 mol%, or 50 to 68 mol%, but are not limited thereto.

[0106] Furthermore, in this invention, based on total liposomes, the liposomes may contain 0.1 to 50 mol%, 0.1 to 45 mol%, 1 to 45 mol%, 5 to 45 mol%, 5 to 40 mol%, 5 to 35 mol%, 5 to 30 mol%, 5 to 25 mol%, 5 to 20 mol%, 5 to 15 mol%, 10 to 40 mol%, 10 to 35 mol%, 10 to 30 mol%, 10 to 25 mol%, 10 to 20 mol%, 20 to 40 mol%, 20 to 35 mol%, 25 to 40 mol%, 25 to 35 mol%, 27 to 32 mol%, 30 to 40 mol%, 5 to 10 mol%, or 40 to 50 mol% cholesterol, but are not limited thereto.

[0107] Furthermore, in this invention, based on total liposomes, it may contain 0.1 to 20 mol%, 0.1 to 15 mol%, 0.1 to 10 mol%, 1 to 20 mol%, 1 to 10 mol%, 1 to 9 mol%, 1 to 8 mol%, 1 to 7 mol%, 1 to 6 mol%, 3 to 20 mol%, 3 to 15 mol%, 3 to 10 mol%, 3 to 7 mol%, 2 to 10 mol%, 3 to 7 mol%, 2 to 6 mol%, 5 to 20 mol%, 5 to 15 mol%, 5 to 10 mol%, 7 to 20 mol%, 7 to 15 mol%, 7 to 12 mol%, 8 to 12 mol%, 9 to 11 mol%, 4 to 6 mol%, or 6 to 8 mol% of lyso-PC, but is not limited thereto.

[0108] In another embodiment of the invention, based on total liposomes, it may contain 0.1 to 50% by dry weight of DSPC.

[0109] Based on total liposomes, it can contain 3 to 50% dry weight of DSPE-mPEG2000.

[0110] Based on total liposomes, it can contain 1 to 80% of dry weight DOPE.

[0111] Based on total liposomes, it can contain cholesterol in amounts ranging from 0.05% to 40% dry weight, and

[0112] Based on total liposomes, it may contain 0.5 to 10% dry weight of lyso-PC, but is not limited thereto.

[0113] In this invention, based on total liposomes, DSPC may be contained in amounts of 0.1 to 50 dry weight%, 0.1 to 45 dry weight%, 0.1 to 40 dry weight%, 1 to 40 dry weight%, 1 to 30 dry weight%, 1 to 20 dry weight%, 1 to 15 dry weight%, 1 to 13 dry weight%, 3 to 20 dry weight%, 5 to 20 dry weight%, 7 to 20 dry weight%, 5 to 15 dry weight%, 7 to 13 dry weight%, or 9 to 11 dry weight%, but is not limited thereto.

[0114] In this invention, based on total liposomes, DSPE-mPEG2000 may be contained in amounts of 3 to 50 dry weight%, 5 to 40 dry weight%, 10 to 30 dry weight%, 10 to 25 dry weight%, 10 to 20 dry weight%, 15 to 30 dry weight%, 15 to 25 dry weight%, 15 to 20 dry weight%, 16 to 19 dry weight%, or 17 to 19 dry weight%, but is not limited thereto.

[0115] In this invention, based on total liposomes, DOPE may be contained in amounts of 1 to 80 dry weight%, 5 to 80 dry weight%, 10 to 80 dry weight%, 15 to 80 dry weight%, 20 to 80 dry weight%, 25 to 80 dry weight%, 30 to 80 dry weight%, 30 to 75 dry weight%, 30 to 70 dry weight%, 30 to 65 dry weight%, 30 to 60 dry weight%, 30 to 55 dry weight%, 30 to 50 dry weight%, 40 to 80 dry weight%, 40 to 70 dry weight%, 50 to 80 dry weight%, 50 to 70 dry weight%, 55 to 65 dry weight%, 60 to 65 dry weight%, 60 to 63 dry weight%, or 60 to 62 dry weight%, but is not limited thereto.

[0116] In this invention, based on total liposomes, the liposomes may contain 0.05 to 40% dry weight, 1 to 40% dry weight, 1 to 30% dry weight, 1 to 20% dry weight, 1 to 15% dry weight, 1 to 13% dry weight, 1 to 10% dry weight, 3 to 20% dry weight, 3 to 15% dry weight, 3 to 13% dry weight, 3 to 10% dry weight, 5 to 20% dry weight, 5 to 15% dry weight, 5 to 13% dry weight, 5 to 10% dry weight, 6 to 8% dry weight, 10 to 40% dry weight, 10 to 35% dry weight, 10 to 30% dry weight, 10 to 25% dry weight, 10 to 20% dry weight, 15 to 40% dry weight, 15 to 30% dry weight, 15 to 25% dry weight, 15 to 22% dry weight, or 20 to 30% cholesterol, but are not limited thereto.

[0117] In this invention, based on total liposomes, the composition may include 0.5 to 10% dry weight, 0.5 to 8% dry weight, 0.5 to 7% dry weight, 0.5 to 6% dry weight, 0.5 to 5% dry weight, 0.5 to 4% dry weight, 1 to 8% dry weight, 1 to 7% dry weight, 1 to 6% dry weight, 1 to 4% dry weight, 2 to 8% dry weight, 2 to 7.5% dry weight, 2 to 6% dry weight, 2 to 5% dry weight, and 2% dry weight. Lyso-PC with dry weights of up to 4%, 2.5% to 7.5%, 2.5% to 7%, 2.5% to 6.5%, 3% to 8%, 3% to 7%, 3% to 6%, 3% to 5%, 3% to 4%, 5% to 10%, 5% to 9%, 5% to 8%, 5% to 7%, 5% to 6%, or 5% to 5.5%, but not limited to these.

[0118] The sonosensitive liposomes according to the present invention contain DOPE (1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine) and sphingolipids as active ingredients, and may not contain DSPC, cholesterol and lyso-PC.

[0119] Preferably, the sonosensitive liposomes according to the present invention contain DOPE, DSPE-mPEG2000 and sphingolipids as active ingredients, and may not contain DSPC, cholesterol and lyso-PC.

[0120] In one embodiment of the invention, based on total liposomes, the DOPE may comprise 5 to 80 mol%, 5 to 75 mol%, 5 to 70 mol%, 5 to 60 mol%, 5 to 50 mol%, 5 to 45 mol%, 5 to 40 mol%, 5 to 35 mol%, 5 to 33 mol%, 5 to 20 mol%, 5 to 10 mol%, 10 to 80 mol%, 10 to 70 mol%, 10 to 60 mol%, 10 to 50 mol%, 20 to 80 mol%, 20 to 70 mol%, 20 to 60 mol%, 20 to 50 mol%, 30 to 80 mol%, 30 to 70 mol%, 30 to 60 mol%, 35 to 55 mol%, or 40 to 55 mol%, but is not limited thereto.

[0121] In another embodiment of the invention, based on total liposomes, the liposomes may comprise 5 to 80 mol%, 5 to 75 mol%, 5 to 70 mol%, 5 to 60 mol%, 5 to 50 mol%, 5 to 45 mol%, 5 to 40 mol%, 5 to 35 mol%, 5 to 20 mol%, 5 to 10 mol%, 10 to 80 mol%, 10 to 70 mol%, 10 to 60 mol%, 10 to 50 mol%, 20 to 80 mol%, 20 to 70 mol%, 20 to 60 mol%, 20 to 50 mol%, 30 to 80 mol%, 30 to 70 mol%, 30 to 60 mol%, 35 to 55 mol%, or 40 to 55 mol%, but are not limited thereto.

[0122] In another embodiment of the invention, DSPE-mPEG2000 may be included in 1 to 20 mol%, 1 to 18 mol%, 1 to 16 mol%, 1 to 14 mol%, 1 to 12 mol%, 5 to 20 mol%, 6 to 20 mol%, 7 to 20 mol%, 8 to 20 mol%, 9 to 20 mol%, 7 to 15 mol%, 7 to 13 mol%, 5 to 15 mol%, 5 to 13 mol%, or 8 to 12 mol%, but is not limited thereto.

[0123] In another embodiment of the invention, the DOPE, sphingolipid, and DSPE-mPEG2000 may be contained in 20-60:20-60:1-20, 20-70:20-70:5-15, 30-60:20-70:5-20, 35-50:35-50:8-15, 25-60:25-60:8-15, or 40-55:40-55:8-15 mol%, but are not limited thereto.

[0124] Furthermore, in this invention, DOPE may be contained in total liposomes at a dry weight percentage of 1 to 80%, sphingolipids may be contained in total liposomes at a dry weight percentage of 1 to 80%, DSPE-mPEG2000 may not be contained, or DSPE-mPEG2000 may be contained in total liposomes at a dry weight percentage of 3 to 50%, but is not limited thereto.

[0125] The liposomes of the present invention are formed from amphiphilic compounds containing phospholipids. Such amphiphilic compounds are typically arranged at the interface between an aqueous medium and a substantially insoluble organic solvent, stabilizing emulsified solvent droplets. The amphiphilic compounds comprise molecules having a hydrophilic polar head (e.g., a polar or ionic group) reactable with an aqueous medium and a hydrophobic organic tail (e.g., a hydrocarbon chain) reactable with, for example, an organic solvent. The amphiphilic compounds are compounds that can stabilize mixtures of substances that are normally immiscible by other methods, such as mixtures of two immiscible liquids (e.g., water and oil), mixtures of liquids and gases (e.g., gaseous microbubbles in water), or mixtures of liquids and insoluble particles (e.g., metal nanoparticles in water).

[0126] Furthermore, the sonosensitive liposomes according to the present invention can be hydrated with ammonium sulfate, ammonium citrate, or TEA-SOS. In other words, the internal buffer of the liposomes can be ammonium sulfate, ammonium citrate, or TEA-SOS. Preferably, the sonosensitive liposomes according to the present invention are hydrated with ammonium sulfate or ammonium citrate.

[0127] The acoustic liposomes according to the invention can penetrate the blood-brain barrier (i.e., pass through or move). In other words, acoustic liposomes can penetrate the blood-brain barrier and enter brain tissue. In particular, the acoustic liposomes according to the invention can penetrate the blood-brain barrier while retaining their physical properties and characteristics. Therefore, the acoustic liposomes according to the invention can be used as carriers for delivering drugs into the brain, i.e., into brain tissue. That is, the compositions according to the invention for penetrating the blood-brain barrier can be used to deliver drugs encapsulated in acoustic liposomes into brain tissue.

[0128] In this invention, the "blood-brain barrier (BBB)" refers to the vascular barrier that separates the brain from the blood, preventing foreign substances such as pigments, drugs, and toxins from entering brain tissue, thereby protecting the brain. The BBB is distributed throughout the cerebral blood vessels surrounding brain cells. The endothelial cells of the brain capillaries are tightly bound to the BBB, and the surrounding glial cells tightly encapsulate it, preventing drugs or metabolites from entering. The substances constituting the BBB are mostly composed of phospholipids.

[0129] When exposed to ultrasound, the liposomes according to the invention cross the blood-brain barrier.

[0130] The term "ultrasound" as used in this invention generally refers to sound waves exceeding the frequencies of audible sound (16 Hz to 20 kHz) that are perceptible to the human ear. High-intensity focused ultrasound (HIFU) is a type of focused ultrasound that delivers continuous, high-intensity ultrasonic energy to a focal point, which, depending on the energy and frequency, can produce transient thermal effects (65-100°C), cavitation effects, mechanical effects, and sonochemical effects. While ultrasound is harmless when passing through human tissue, the high-intensity ultrasound at the focal point can generate sufficient energy to cause coagulative necrosis and thermal ablation effects, regardless of tissue type.

[0131] In this invention, the liposomes are acoustic liposomes. Acoustic liposomes can refer to liposomes whose permeability increases upon exposure to ultrasound. Therefore, when liposomes are exposed to ultrasound, the drug loaded within them can be released. Alternatively, acoustic liposomes can refer to liposomes whose ability to penetrate the blood-brain barrier increases upon exposure to ultrasound. Therefore, when liposomes are exposed to ultrasound, they can cross the blood-brain barrier and be delivered to the brain; similarly, the drug encapsulated in the liposomes can also be delivered to the brain via ultrasound.

[0132] In this invention, ultrasound refers to sound waves with frequencies greater than 16 Hz to 20 kHz in the audible frequency range. Ultrasound can be high-intensity focused ultrasound (HIFU), high-intensity unfocused ultrasound, or a combination of both, but is not limited thereto. HIFU refers to ultrasound that concentrates high-intensity ultrasound energy to form a focal point. Depending on the image viewed during high-intensity focused ultrasound processing, there are ultrasound-guided HIFU and magnetic resonance imaging-guided HIFU.The frequency of ultrasound can be, for example, 1 kHz to 100 kHz, 1 kHz to 90 kHz, 1 kHz to 80 kHz, 1 kHz to 70 kHz, 1 kHz to 60 kHz, 1 kHz to 50 kHz, 1 kHz to 40 kHz, 1 kHz to 30 kHz, 1 kHz to 20 kHz, 1 kHz to 10 kHz, 20 kHz to 3.0 MHz, 40 kHz to 2.0 MHz, 60 kHz to 2.0 MHz, 80 kHz to 2.0 MHz, 100 kHz to 2.0 MHz, 150 kHz to 2.0 MHz, 200 kHz to 2.0 MHz, 250 kHz. Hz to 2.0MHz, 300kHz to 2.0MHz, 350kHz to 2.0MHz, 400kHz to 2.0MHz, 450kHz to 2.0MHz, 500kHz to 2.0MHz, 550kHz to 2.0MHz, 600kHz to 2.0MHz, 650kHz to 2.0MHz, 700kHz to 2.0MHz, 750kHz to 2.0MHz, 800kHz to 2.0MHz, 850kHz to 2.0MHz, 900kHz to 2.0MHz, 950kHz to 2.0MHz, 600kHz to 1.5MHz 650kHz to 1.5MHz, 700kHz to 1.5MHz, 750kHz to 1.5MHz, 800kHz to 1.5MHz, 850kHz to 1.5MHz, 900kHz to 1.5MHz, 950kHz to 1.5MHz, 1MHz to 1.5MHz, 600kHz to 1.3MHz, 650kHz to 1.3MHz, 700kHz to 1.3MHz, 750kHz to 1.3MHz, 800kHz to 1.3MHz, 850kHz to 1.3MHz, 900kHz to 1.3MHz, 950kHz to 1.3MHz, 950kHz to 1.3MHz. MHz, 600kHz to 1.1MHz, 650kHz to 1.1MHz, 700kHz to 1.1MHz, 750kHz to 1.1MHz, 800kHz to 1.1MHz, 850kHz to 1.1MHz, 900kHz to 1.1MHz, 950kHz to 1.1MHz, 600kHz to 1MHz, 650kHz to 1MHz, 700kHz to 1MHz, 750kHz to 1MHz, 800kHz to 1MHz, 850kHz to 1MHz, 900kHz to 1MHz, or 950kHz to 1MHz, but not limited to these.

[0133] This ensures the stability of the liposomes. Stability refers to the condition where the drug encapsulated in the liposomes is not released into the bloodstream without exposure to ultrasound. The inventors of this invention have demonstrated that, when the release rate of the drug encapsulated in the acoustic liposomes according to the invention is measured every 20 minutes under vortex conditions at room temperature (20–25°C) for 60 minutes, a maximum drug release rate of 30% or less was observed. In other words, the liposomes according to the invention maintain a stable structure without ultrasound, and therefore the drug is hardly released.

[0134] The particle size of the liposomes according to the present invention can be, for example, 50 nm to 500 nm, 50 nm to 400 nm, 50 nm to 300 nm, 50 nm to 200 nm, 60 nm to 200 nm, 70 nm to 200 nm, 80 nm to 200 nm, 90 nm to 200 nm, 100 nm to 200 nm, 110 nm to 190 nm, 120 nm to 180 nm, 130 nm to 170 nm, 140 nm to 170 nm, 140 nm to 160 nm, or about 150 nm. According to a specific embodiment, it can be 100 nm to 200 nm. The particle size distribution of the liposomes can be measured using a Zetasizer Nano ZS (Malvern) by dynamic light scattering (DLS) analysis after diluting the liposomes 10-fold. However, the method for measuring the particle size distribution of liposomes is not limited to this, and can be measured according to other methods disclosed in the art, and can be converted to equivalent values.

[0135] The acoustic liposomes according to the present invention can encapsulate drugs. The term "encapsulation" may be used interchangeably with the terms "incorporation" or "loading".

[0136] As used in this invention, the term "medicine" refers to any compound having the desired biological activity. Desired biological activity includes activities that can be used to diagnose, cure, alleviate, treat, or prevent diseases in humans or other animals.

[0137] In this invention, the mixing ratio of the drug to the sonosensitive liposomes is preferably 1:2 to 1:50 by mass (w / w%), more preferably 1:2 to 1:40, 1:2 to 1:35, 1:2 to 1:30, 1:2 to 1:25, 1:10 to 1:50, 1:10 to 1:40, 1:10 to 1:35, 1:10 to 1:30, 1:10 to 1:35, 1:2 to 1:20, 1:2 to 1:18, 1:2 to 1:16, 1:2 to 1:14, 1:2 to 1:12, 1:2 to 1:1 0.5, 1:2.5 to 1:10.5, 1:2.5 to 1:5, 1:2 to 1:4, 1:2.5 to 1:4.5, 1:2.5 to 1:4, 1:2.5 to 1:3, 1:1:5 to 1:10.5, 1:5.5 to 1:10.5, 1:6 to 1:10.5, 1:6.5 to 1:10.5, 1:7 to 1:10.5, 1:7 to 1:10, 1:7 to 1:9, or about 1:8 mass ratio (w / w%), but not limited to these, as long as the drug can be effectively incorporated into the liposomes.

[0138] When a drug is treated with liposomes to encapsulate it into liposomes according to the invention, the proportion of the drug encapsulated in the liposomes to the total added drug can be 50% to 100%. That is, the drug encapsulation rate of the liposomes according to the invention can be 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 76% to 100%, 77% to 100%, 80% to 100%, 85% to 100%, 87% to 100%, 88% to 100%, 90% to 100%, or 95% to 100%, but is not limited thereto.

[0139] Encapsulation efficiency can refer to the loading rate of the added drug amount, but is not limited to this. Methods for measuring drug encapsulation efficiency in liposomes include treating the drug with liposomes, separating the unencapsulated drug using size exclusion chromatography (SEC), and calculating it by measuring the absorbance of the encapsulated and unencapsulated drugs. However, methods for measuring drug encapsulation efficiency are not limited to this; it can be measured according to other methods disclosed in the art and can be converted to values ​​at equivalent levels.

[0140] In this invention, the drug release rate of the acoustic liposomes encapsulating the drug can be 10% to 100%, 50% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, or 75% to 100%, but is not limited thereto. The drug release rate can refer to the release rate of the added drug amount, but is not limited thereto. Here, the method for measuring the release rate includes sonicating the liposomes, separating the drug released from the liposomes and the liposomes from which the drug has been released using the SEC method, and quantifying it by measuring the absorbance of the drug. However, the method for calculating the drug release rate is not limited thereto; it can be measured according to other methods disclosed in the art and can be converted to values ​​at equivalent levels.

[0141] In this invention, the drug release rate of the sonosensitive liposomes encapsulating the drug in the bloodstream under untreated ultrasound conditions is 0.1% to 100%, 0.1% to 80%, 0.1% to 60%, 0.1% to 40%, 0.1% to 30%, 0.1% to 20%, 0.1% to 10%, or less than 30%, but is not limited thereto.

[0142] The compositions according to the invention can be used for the purpose of delivering drugs across the blood-brain barrier to the brain. Specifically, the invention provides a blood-brain barrier-penetrating drug delivery carrier comprising, as an active ingredient, a sonosensitive liposome according to the invention.

[0143] In this invention, "medicine" can include, but is not limited to, any medicine that acts in the brain. That is, medicine can include, but is not limited to, any medicine intended to be delivered to the brain, regardless of its specific type or composition. Preferably, medicine can be a therapeutic agent for brain diseases.

[0144] In this invention, "brain disease" refers to a pathological state in which damage to brain tissue, blood vessels, and nerves leads to a reversible or irreversible reduction or loss of brain tissue function or structure. Preferably, brain disease can be selected from the group consisting of: brain tumors, brain infections caused by bacteria or viruses, Parkinson's disease, encephalitis, stroke, paralysis, Alzheimer's disease, Lujarig's disease, Huntington's disease, Pick's disease, Kreutzfeldt-Jacob's disease, epilepsy, thrombosis, embolism, cerebral infarction, paralysis, small artery occlusion, and brain metabolic disorders, but is not limited thereto, and includes all diseases related to the brain.

[0145] Most preferably, the brain disease can be a brain tumor (brain cancer). Preferably, the brain tumor can be selected from the group consisting of: astrocytoma, glioma, brainstem glioma, pituitary adenoma, glioblastoma, oligodendroglioma, glioblastoma multiforme, oligodendroglioma, oligoastrocytoma, ependymoma, medulloblastoma, hemangioblastoma, meningioma, pituitary adenoma, craniopharyngioma, and choroid plexus papilloma, but is not limited thereto.

[0146] As used in this article, the term "cancer" or "tumor" refers to a disease caused by cells that disregard normal growth limits and exhibit aggressive characteristics of division and growth, invasive characteristics of invading surrounding tissues, and metastatic characteristics of spreading to other parts of the body.

[0147] The term "brain disease treatment agent" may include at least one selected from the group consisting of: vincristine, vinblastine, vinflunidine, vinorelbine, temozolomide, carmustine, lomustine, cabazitaxel, docetaxel, larotaxel, oxalitaxel, paclitaxel, testataxel, ixapril, lomustine, procarbazine, rituximab, tocilizumab, temozolomide, carboplatin, erlotinib, irinotecan, enzartolin, vorinostatin, doxorubicin, cisplatin, glimepiride, and 5-fluorouracil. Tamoxifen, topotecan, belotecone, imatinib, fluorouracil, gemcitabine, leuprorelin, flutamide, zoledronic acid, methotrexate, camptothecin, hydroxyurea, streptozocin, penoxuridine, retinic acid, nitrogen mustard, chlorambucil, busulfan, doxyfluridine, mitomycin, prednisone, everolimus, mitoxantrone, levodopa, carbidopa, entacapone, tocapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics, and amantadine, but not limited to these.

[0148] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of brain diseases, comprising, according to the invention, acoustic liposomes as an active ingredient. Preferably, the acoustic liposomes encapsulate the brain disease therapeutic agent.

[0149] In this invention, "prevention" refers to all the effects of inhibiting or delaying the onset of brain diseases by applying the composition according to the invention.

[0150] In this invention, "treatment" refers to all effects of improving or advantageously altering the symptoms of brain diseases by applying the composition according to the invention.

[0151] As used herein, the term "pharmaceutical composition" refers to a substance manufactured for the purpose of preventing or treating brain diseases and formulated in various forms according to conventional methods. For example, it may be formulated into oral forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups; or into topical forms such as creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes, or poultices; or into forms such as suppositories and sterile injectable solutions.

[0152] In addition to the acoustic liposomes according to the invention, the composition may also contain at least one selected from the group consisting of anticancer agents, imaging contrast agents, antibiotics, anti-inflammatory agents, proteins, cytokines, peptides and antibodies.

[0153] Furthermore, the pharmaceutical compositions according to the invention may also contain suitable carriers, excipients, and diluents conventionally used in the preparation of pharmaceutical compositions. As carriers, excipients, and diluents that may be included in the pharmaceutical compositions according to the invention, lactose, dextran, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil may be used. For formulations, commonly used diluents or excipients, such as fillers, thickeners, binders, wetting agents, disintegrants, and surfactants, are used. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., which are formulated by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants, such as magnesium stearate and talc, are also used. Examples of liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups. These liquid formulations, in addition to simple and commonly used diluents such as water and liquid paraffin, can contain various types of excipients, such as wetting agents, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-limiting examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. For suppository bases, substances such as Witepsol, polyethylene glycol, Tween 61, cocoa butter, laurin butter, and glycerin gelatin can be used.

[0154] The pharmaceutical compositions of the present invention can be administered to subjects via a variety of routes. All methods of administration are predictable, and the pharmaceutical compositions can be administered, for example, by oral administration, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, intrathecal (perispinal space) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, intraauricular administration, intranasal administration, inhalation, oral or nasal spray, transdermal administration, and transdermal administration.

[0155] The pharmaceutical compositions according to the invention are administered in a pharmaceutically effective amount. In this invention, a "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the effective dose level can be determined based on factors including the patient's disease type, disease severity, drug activity, drug sensitivity, time of administration, route of administration, excretion rate, treatment duration, and concurrent drug use, as well as other factors well known in the medical field.

[0156] The compositions according to the invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with therapeutic agents in related fields, and can be administered in single or multiple doses. Taking all the foregoing factors into account, it is important to administer the composition in the minimum amount necessary to achieve maximum effect without any side effects, and this can be readily determined by those skilled in the art. Administration can be performed once daily or divided into several doses throughout the day.

[0157] As used herein, “application” means providing a subject with the predetermined composition of the present invention by any suitable method.

[0158] The term "subject" as used in this article refers to a subject who needs treatment for a disease, and more specifically, mammals such as humans or non-human primates, mice, dogs, cats, horses, and cattle.

[0159] The compositions according to the invention can be administered sequentially or simultaneously with ultrasound treatment. Preferably, the compositions can be administered immediately after ultrasound treatment. In particular, ultrasound can be applied to the brain. The inventors of the invention have identified safe ultrasound parameters capable of opening the blood-brain barrier without causing damage to brain tissue, and the efficiency of drug delivery to the brain by liposomes according to the invention can be further enhanced when the pharmaceutical composition is administered simultaneously with ultrasound under these conditions.

[0160] Preferably, the frequency of the ultrasound can be 20 kHz to 3 MHz, 20 kHz to 2 MHz, 20 kHz to 1.5 MHz or 20 kHz to 1 MHz, but is not limited thereto.

[0161] Preferably, the intensity of the ultrasound can be 0.1 to 5W, 0.1 to 4W, 0.1 to 3W, 0.1 to 2W, 0.1 to 1.5W, 0.5 to 3W, 0.5 to 2W, or 0.7 to 1.5W, but is not limited thereto.

[0162] In addition, the duty cycle of ultrasound can be 0.5% to 20%, 0.5% to 15%, 0.5% to 12%, 0.5% to 10%, 0.5% to 7%, 0.7% to 10%, 0.8% to 10%, 0.9% to 10%, 1% to 10%, 1% to 9%, 1% to 8%, 1% to 7%, 1% to 6%, 1% to 5%, 1% to 4%, 1% to 3%, 2% to 10%, 3% to 10%, or 4% to 10%, but is not limited to these.

[0163] In addition, ultrasound can be emitted for 10 to 300 seconds, 10 to 250 seconds, 10 to 200 seconds, 10 to 150 seconds, 10 to 120 seconds, 10 to 100 seconds, 10 to 90 seconds, 10 to 80 seconds, 10 to 70 seconds, 20 to 100 seconds, 30 to 100 seconds, 40 to 100 seconds, 50 to 100 seconds, 50 to 90 seconds, 50 to 80 seconds, or 50 to 70 seconds, but is not limited to these.

[0164] Furthermore, ultrasound treatment can be performed sequentially with or simultaneously with the application of microbubbles. Preferably, ultrasound treatment can be performed immediately after the application of microbubbles. In this invention, microbubbles refer to bubbles with an average size of 1 to 10 μm that serve as ultrasound contrast agents, inducing cavitation and temporarily opening the blood-brain barrier together with ultrasound.

[0165] Preferably, based on the subject's total body weight, the microbubbles can be administered in the following amounts: 10 to 1 × 10⁻⁶. 10 Particles / g, 10 to 1×10 9 Particles / g, 10 to 1×10 8 Particles / g, 10 to 1×10 7 Particles / g, 10 to 1×10 6 Particles / g, 10 to 1×10 5 Particles / g, 10×10 2 Up to 1×10 8 Particles / g, 10×10 3 Up to 1×10 8 Particles / g, 10×10 4 Up to 1×10 8 Particles / g, 1×10 6 Up to 9×10 6 Particles / g, 1×10 6 Up to 8×10 6 Particles / g, 1×10 6 Up to 7×10 6 Particles / g, 1×10 6 Up to 6×10 6 Particles / g, 2×10 6 Up to 7×10 6 Particles / g, or 4×10 6 Up to 7×10 6 Particles / g, but not limited to this.

[0166] Furthermore, the present invention provides a method for generating acoustic liposomes, comprising the following steps:

[0167] (S1) Dissolve at least one selected from the group consisting of DSPC, DSPE-mPEG2000, DOPE, cholesterol and lyso-PC in a first organic solvent;

[0168] (S2) Evaporating organic solvents to manufacture liposome membranes; and

[0169] (S3) Hydrate the liposome membrane with an aqueous solution.

[0170] In this invention, the first organic solvent may be at least one selected from the group consisting of: dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and diethyl ether, but is not limited thereto.

[0171] In this invention, the aqueous solution can be ammonium sulfate, ammonium citrate, or TEA-SOS, but is not limited thereto, and can be appropriately selected according to the type of drug to be encapsulated in the liposomes. Most preferably, the aqueous solution is ammonium sulfate.

[0172] In this invention, the first organic solvent in step (S1) may contain polysorbate, but is not limited thereto.

[0173] The manufacturing method may also include the step of extruding the hydrated liposomes after step (S3) using an extruder. The extrusion temperature of the liposomes can be adjusted in various ways from room temperature to the transition temperature range of each material, and the extrusion can be repeated an appropriate number of times to ensure uniform liposome size.

[0174] Furthermore, the present invention provides a method for encapsulating a brain disease therapeutic agent in a sonic liposome for penetrating the blood-brain barrier, the method comprising the step of mixing the brain disease therapeutic agent with the sonic liposome.

[0175] Preferably, the mixing step can be carried out at 25 to 70°C, 30 to 70°C, 40 to 70°C, 50 to 70°C, 55 to 70°C, 50 to 65°C, or 55 to 65°C.

[0176] In addition, the mixing steps can be carried out for 30 minutes to 5 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 1 hour to 5 hours, 1 hour to 4 hours, or 1 hour to 3 hours.

[0177] In considering their function in this invention, the terminology used herein is selected from commonly used terms that are currently widely used. However, these may vary depending on the intent of those skilled in the art, precedent, the emergence of new technologies, etc. Furthermore, in some cases, there are terms arbitrarily chosen by the applicant; in such cases, the meanings of these terms will be explained in detail in the description of the corresponding invention. Therefore, the terminology used herein should not be defined solely by its name, but rather based on its meaning and the overall content of the invention.

[0178] Throughout this specification, when a part is referred to as "comprising (including)" a component, it does not exclude other components, but may include other components, unless otherwise expressly stated. Degree terms such as "about" and "substantially" used throughout this specification are used in a meaning close to or equal to numbers when referring to manufacturing and material tolerances inherent in the foregoing meaning, and are intended to prevent unscrupulous infringers from unfairly exploiting this disclosure, wherein precise or absolute numerical values ​​are mentioned to aid in understanding the invention.

[0179] Throughout this specification, the term "combination thereof" included in the Markush formulation refers to a mixture or combination of at least one of the groups consisting of the components listed in the Markush formulation, which means that it includes at least one of the groups consisting of these components.

[0180] Preferred embodiments are provided below to aid in understanding the present invention. However, the following embodiments are provided only to aid in understanding the present invention, and the scope of the present invention is not limited to the following embodiments.

[0181] [Example]

[0182] The materials used to manufacture the acoustic liposomes for penetrating the blood-brain barrier according to the present invention are listed in the table below.

[0183]

[0184] Example 1. Manufacturing of liposomes for penetrating the blood-brain barrier and drug delivery, and encapsulation of drugs.

[0185] A sonosensitive liposome (IMP302) optimized for brain tumor treatment was fabricated. Initially, to evaluate the effect of solvent on drug loading efficiency, liposomes with the lipid compositions shown in Table 1 were prepared and experiments were conducted.

[0186] [Table 1]

[0187]

[0188] 1-1. Fabrication and Hydration of Lipid Membranes

[0189] The total lipid content for each sample was set at 32 mg, and all of it was dissolved in 2 ml of chloroform. Lipid films were generated by completely evaporating the chloroform using a rotary evaporator. Subsequently, each lipid film was hydrated using 250 mM ammonium sulfate, ammonium citrate, or TEA-SOS as an internal buffer. Hydration was achieved at a lipid concentration of 32 mg / mL by stirring the lipid films and each solution at 50 °C and 200 rpm.

[0190] 1-2. Size control of liposomes using size extrusion

[0191] The liposomes produced via 1-1 extrusion exhibit a multilayered structure and a polydisperse size distribution. To convert the liposomes into a monolayer structure and control the size to 100–200 nm, a sizeextruder mini (Avanti) equipped with a polycarbonate filter was used. Size extrusion was controlled using a polycarbonate filter with a pore size of 200 nm. Ten to twenty reciprocating extrusions were performed using a syringe to obtain monodisperse liposomes.

[0192] 1-3. Exchange of external buffer solution

[0193] To induce remote loading, the external buffer of the manufactured liposomes was replaced with deionized water (DW) using a PD-10 column. Liposomes with the external buffer replaced by deionized water were obtained by loading 2 mL of liposome solution into a PD-10 column and then eluting with 4 mL of DW.

[0194] 1-4. Packaging of Vincristine Sulfuric Acid

[0195] To encapsulate vincristine sulfate into the obtained liposomes, vincristine sulfate and lipids were mixed in deionized water at a ratio of 1:20 (w / w%), and then stirred at 150 rpm for 2 hours at 37°C or 60°C. Subsequently, unencapsulated vincristine sulfate was removed using a PD-10 column in the same manner as described above. The loading efficiency of vincristine in the liposomes was quantitatively analyzed by measuring absorbance at 294 nm using UV-vis.

[0196] 1-5. Characterization and Optimization of Preparation Methods for Acoustic Liposomes Used to Penetrate the Blood-Brain Barrier

[0197] Acoustic liposomes for penetrating the blood-brain barrier were obtained by the lipid film hydration method according to the above embodiments. To verify the characteristics of liposomes based on the type of hydration solution used in liposome manufacturing, the size distribution of the liposomes and the encapsulation efficiency of vincristine sulfate were compared according to the type of hydration solution. The results are shown in Table 2 below.

[0198] [Table 2]

[0199]

[0200] As shown in Table 2, the size distribution of the acoustic liposomes for blood-brain barrier penetration fabricated using each internal buffer solution exhibits a size of approximately 200 nm. Furthermore, the encapsulation efficiency (w / w%) of vincristine sulfate for each liposome shows a level corresponding to that of the commercial liposome formulation Marqibo.

[0201] Furthermore, to determine the optimal conditions for loading vincristine sulfate into sonosensitive liposomes manufactured with ammonium citrate for blood-brain barrier penetration, a comparative evaluation was performed using UV-vis to measure loading efficiency based on temperature and time during the vincristine sulfate and liposome mixing phase. The loading effects for each temperature or time condition are as follows.

[0202] [Table 3]

[0203]

[0204] The loading efficiency of vincristine sulfate varied with temperature, showing 3.5% at 37°C and 4.7% at 60°C. Therefore, loading vincristine sulfate at 60°C demonstrated that it was more efficient in terms of loading efficiency. Furthermore, when comparing loading times of 2 hours and 24 hours, both showed similar loading efficiencies. The results also revealed that the size distribution of liposomes remained stable and unchanged during the loading process of vincristine sulfate.

[0205] This embodiment ultimately confirms that using ammonium citrate (pH 3.2) or ammonium sulfate (pH 6.4) as internal buffers can most effectively manufacture acoustic liposomes loaded with vincristine sulfate for crossing the blood-brain barrier. For even more efficient loading, optimal conditions were found involving loading the therapeutic agent onto the liposomes at 60°C for 2 hours.

[0206] Example 2. Comparison of the properties of acoustic liposomes for penetrating the blood-brain barrier based on lipid composition.

[0207] To determine the optimal composition of acoustic liposomes for treating brain tumors, we compared their physical properties and characteristics based on the types of lipids that make up the liposomes. The compositions of the liposomes used in the comparative experiments are shown in Table 4 below.

[0208] [Table 4]

[0209]

[0210]

[0211] Each liposome was manufactured according to the method of Example 1, but doxorubicin hydrochloride, which has a higher loading efficiency than vincristine, was used for clear comparison. Formulations 004P and 013P, comprising polysorbate 80, were prepared by adding 0.1% (v / v) of polysorbate 80 to a solvent.

[0212] 2-1. Encapsulation and Encapsulation Efficiency Analysis of Doxorubicin Hydrochloride

[0213] Doxorubicin hydrochloride was encapsulated by mixing it with liposomes at a weight ratio of 1:8 and then stirring at 150 rpm for 2 hours at 37°C. Unloaded doxorubicin hydrochloride was removed using a PD-10 column. The doxorubicin hydrochloride loaded into the liposomes was quantified by analyzing the absorbance at 475 nm using UV-vis.

[0214] Candidate compositions for acoustic liposomes for treating brain tumors were initially screened through basic physical property analysis, and the optimal composition was determined by drug release assays using ultrasound. The component size distribution and the effect of doxorubicin hydrochloride encapsulation in acoustic liposomes for penetrating the blood-brain barrier are shown in Table 5 below.

[0215] [Table 5]

[0216]

[0217]

[0218] According to Table 5, each liposome prepared with the composition exhibited a size distribution of 100 to 150 nm, showing a polydispersity index of 0.1, indicating a uniform size distribution. Furthermore, the doxorubicin hydrochloride loading efficiency showed excellent loading rates, with over 90% of the added doxorubicin volume loaded into the liposomes. The exception was composition 008, which exhibited unstable particles during the manufacturing phase, agglomerating rapidly and tending to aggregate further over time. However, in the other compositions, no particle aggregation or drug leakage occurred over time. To select the optimal composition for liposomes, compositions 004, 005, and 013 were chosen as candidate compositions, which are expected to exhibit excellent ultrasonic responsiveness and long cycling performance due to their high doxorubicin encapsulation efficiency and high DOPE content.

[0219] 2-2. Analysis of the drug release effect by ultrasound

[0220] The ultrasonic response of liposomes was tested using compositions 004, 005, and 013, which were selected as candidate compositions. Analysis of drug release rate by ultrasound was performed using an ultrasound generator, in which liposomes containing the drug were placed in the ultrasound generator and subjected to an ultrasound at a frequency of 24 kHz and an amplitude of 20% (92 W / cm²). 2 The intensity was increased by sonication for 60 seconds. Drug released from the liposomes was separated by a PD-10 column and quantified by measuring absorbance at 295 nm using UV-vis. As experimental controls, composition 011 containing relatively low acoustic sensitivity lipids and Doxil composition liposomes loaded with vincristine sulfate or doxorubicin hydrochloride (commercial products) were used.

[0221] The loading rates of vincristine sulfate or doxorubicin hydrochloride for each liposome and the drug release rates via ultrasound are shown in Table 6 below.

[0222] [Table 6]

[0223]

[0224] First, comparing loading efficiencies, compositions 004, 005, 011, and Doxil exhibited high loading efficiencies for doxorubicin hydrochloride, with over 90% of the added drug encapsulated. Only in the case of composition 013 was a slightly lower loading efficiency of 51.8% observed due to liposome aggregation during the loading process of doxorubicin hydrochloride. However, for vincristine sulfate, all compositions showed high loading efficiencies of 70% to 80%.

[0225] Drug release due to ultrasound responsiveness was assessed by quantifying the amount of drug released from each liposome based on ultrasound stimulation. For liposomes containing doxorubicin hydrochloride, it was confirmed that the drug release rate due to ultrasound responsiveness increased in the order of higher DOPE composition; therefore, the DOPE composition ratio was proportional to the ultrasound responsiveness of the liposome. For sonosensitive liposomes containing vincristine sulfate, most liposomes exhibited similar levels of ultrasound responsiveness under the same ultrasound conditions.

[0226] Therefore, based on these experimental results, vincristine, a drug for treating brain tumors, was found to have the highest loading efficiency, and the liposomes of compositions 004 and 013 were identified as the optimal liposome compositions, exhibiting excellent performance in terms of physical properties, drug encapsulation efficiency, and ultrasonic responsiveness when loading doxorubicin. Particularly in the case of composition 013, although the loading efficiency of doxorubicin was slightly lower, it was included in the optimal liposome composition because it exhibited a high loading efficiency similar to other vincristine compositions.

[0227] 2-3. Confirm the effect of polysorbate 80 on the ultrasound responsiveness of liposomes.

[0228] In addition, to evaluate the effect of polysorbate 80 on sonic liposomes used for penetrating the blood-brain barrier, the physical properties and sonic responsiveness of liposomes from compositions 004 and 013, which exhibited the highest release of vincristine sulfate and doxorubicin hydrochloride, were assessed after the addition of polysorbate 80. In this invention, polysorbate 80 is added as a component to increase the BBB penetration rate of the liposome formulation. The liposomes were manufactured using a solvent containing 0.1% (v / v) polysorbate 80, with all other processes performed identically. The physical properties of the liposomes containing polysorbate 80 and the release rate of doxorubicin hydrochloride due to sonic responsiveness are shown in Table 7 below.

[0229] [Table 7]

[0230] Size (d.nm) 128.2 146.2 Loading efficiency (%) 95.3 94.1 release(%) 73.9 65.9

[0231] As shown in Table 7, when polysorbate 80 was added to the compositions of liposomes 004 and 013, the particle size distributions were 128.2 nm and 146.2 nm, respectively, and the drug release rates induced by sonic responsiveness were 73.9% and 65.9%, respectively. These experimental results indicate that polysorbate 80 does not affect the physical properties of the liposomes before it promotes the penetration of sonic liposomes across the blood-brain barrier, as the addition of polysorbate 80 enhances the BBB penetration rate of the liposomes.

[0232] Example 3. Evaluation of the brain tumor cell penetration ability of acoustic liposomes used to penetrate the blood-brain barrier by FACS analysis.

[0233] Experiments were conducted using the U87MG brain tumor cell line to evaluate the ability of liposomes to infiltrate brain tumor cell lines according to their composition. To quantitatively analyze the cell line penetration effect of each liposome, FACS analysis was performed. The acoustic liposomes used in the experiments for penetrating the blood-brain barrier were labeled with the fluorescent dye DiI and detected in the red wavelength range. The DiI dye exhibited fluorescence at excitation / emission wavelengths of 549 / 565 nm in the red wavelength range.

[0234] The experiments were conducted as follows. Before liposome treatment, the U87MG cell line was starved in serum-free cell culture medium for 1 hour. The starved cells were then treated with each liposome solution at a concentration of 400 μg / ml, and incubated with the liposomes for 2 or 4 hours before FACS analysis. The cell number used was 3 × 10⁶ cells / ml. 5 Quantitative analysis results of the brain tumor cell infiltration ability of liposomes are as follows: Figure 1 As shown. The liposomes 004 and 013, as well as the Doxil composition, which are acoustic liposomes according to the present invention for penetrating the blood-brain barrier, exhibited high endocytosis efficiency; most liposomes infiltrated the U87MG cells within 2 hours after treatment. The penetration efficiency of U87MG cells was shown in the order of Doxil, 004, 013, and 011 liposomes.

[0235] Furthermore, when brain tumor cells and liposomes were incubated for 4 hours, the liposomes of compositions 004, 013, and Doxil exhibited near 100% infiltration efficiency, with cell infiltration rates increasing over time. The liposomes of composition 011 also showed a sustained increase in endocytosis efficiency over time. In particular, the brain tumor cell infiltration effect of the acoustic liposomes according to the present invention was more than twice that of the commercially available liposome Marqibo used for loading vincristine.

[0236] These results demonstrate that the acoustic liposomes according to the invention for penetrating the blood-brain barrier can effectively penetrate brain tumor cells and deliver drugs.

[0237] Example 4. Verification of the cell penetration ability of acoustic liposomes for penetrating the blood-brain barrier using confocal fluorescence microscopy.

[0238] To select the optimal liposome composition for brain tumor treatment, confocal fluorescence microscopy was used to evaluate the cell infiltration effects of different liposome compositions on the U87MG brain tumor cell line over time after treatment. The intracellular location of each liposome was detected by labeling it with DiI fluorescent dye and irradiating it with red wavelength. U87MG cells were seeded into a cell culture chamber, incubated overnight, and starved with serum-free medium for 1 hour before each liposome treatment. Each liposome was used to treat the starved cells at a concentration of 400 μg / ml, and the particles were washed after inducing cell uptake by incubation for 2 or 4 hours. Subsequently, for confocal microscopy observation, the cells were fixed with 4% paraformaldehyde and then observed under a microscope. Imaging was performed using confocal fluorescence microscopy to analyze the degree and process of brain tumor cell infiltration based on the liposome composition. For fluorescence observation, DiI-labeled liposomes were photographed at red wavelengths (ex / em: 549 / 565 nm), and cell nuclei were stained blue with DAPI (ex / em: 358 / 461 nm). Cell morphology was confirmed using DIC optical imaging. Observations of U87MG cell infiltration over time using compositions 004 and 013 according to the invention, as well as liposomes of Doxil, showed… Figure 2a and 2b middle.

[0239] Confocal fluorescence microscopy was used to observe the infiltration effect of each liposome particle in brain tumor cells over time. It was found that liposomes from compositions 004 and 013 according to the present invention effectively infiltrated U87MG cells within 2 hours, consistent with the FACS analysis results of Example 3. All liposomes of each composition were accurately located within the cytoplasm of U87MG cells, exhibiting punctate fluorescence, indicating that the liposomes underwent endocytosis while maintaining their particle morphology. No significant changes were observed in the morphology of U87MG cells treated with the liposomes according to the present invention or the Doxil composition liposomes compared to the untreated control group. These results indicate that the sonosensitive liposomes of the present invention have very low cytotoxicity.

[0240] Example 5. Validation of the brain tumor cell killing effect of acoustic liposomes used to penetrate the blood-brain barrier.

[0241] To verify the anticancer effect of the acoustic liposomes according to the present invention, U87MG brain tumor cells were used to verify the anticancer effect. U87MG cells were cultured at 5 × 10⁶ cells per well.4 Cells were seeded into 96-well plates and cultured for 24 hours. Dürbeco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics was used as the cell culture medium. To assess efficacy, liposomes loaded with doxorubicin composition 004, Doxil composition liposomes, and free doxorubicin were added to the cells at different concentrations (0, 1.25, 2.5, 5, 10 μg / mL). To compare the degree of cancer cell death depending on the presence or absence of ultrasound irradiation, liposomes irradiated with ultrasound and those not irradiated were treated in designated wells. After drug treatment, the cells were incubated for 4 hours, and after removing all added drugs, they were washed three times with phosphate-buffered saline (0.01 M, pH 7.4). Cancer cell proliferation was compared by culturing in the above cell culture medium for 72 hours. The anti-cancer cell killing effect was quantitatively analyzed by MTT assay and measurement of absorbance at 570 nm.

[0242] The verification results of the anti-cancer cell killing effects of various liposomes are as follows: Figure 3 As shown. In the case of the acoustic liposomes according to the invention for penetrating the blood-brain barrier, the anti-cancer cell killing effect was significantly enhanced after ultrasound treatment compared with that before ultrasound treatment. On the other hand, in the case of Doxil, which does not respond to ultrasound, there was no difference in the anti-cancer cell killing effect before and after ultrasound treatment. In particular, the liposomes of composition 004 according to the invention showed an anti-cancer effect more than twice that of Doxil liposomes. These results indicate that the acoustic liposomes according to the invention for penetrating the blood-brain barrier have excellent anti-cancer activity against brain tumor cells.

[0243] Example 6. Exploration of microbubble conditions for blood-brain barrier opening and comparative analysis of Evans blue penetration efficiency

[0244] To determine the optimal ultrasound conditions for opening the blood-brain barrier (BBB), the degree of BBB opening was compared and analyzed using Evans blue. Evans blue is a very small dye with a molecular weight of 961 Da, widely used for analyzing the degree of BBB opening. Experiments were conducted using normal BALB / c nude mice, and ultrasound was emitted using a VIFU2000 high-intensity focused ultrasound device after intravenous injection of microbubbles. Immediately after the BBB was temporarily opened by ultrasound and microbubble cavitation, Evans blue (3%, w / w) was injected intravenously, and the brain was extracted 24 hours later. Prior to brain extraction, blood was drained by perfusion with physiological saline to eliminate potential blood-related variables. Brain tissue was immersed in formamide and incubated overnight at 70°C to extract Evans blue. The degree of BBB opening was quantitatively analyzed by measuring absorbance at wavelengths of 670 nm and 740 nm using UV-Vis.

[0245] Factors influencing ultrasound and microbubble cavitation (a mechanism for opening the blood-brain barrier) include ultrasound conditions such as frequency, intensity, duty cycle, and emission time, as well as the number of microbubbles. Therefore, to determine the ultrasound conditions and microbubble number required to open the blood-brain barrier, the degree of barrier opening was first quantitatively analyzed based on the number of microbubbles. Table 8 below shows the comparative experimental design of the degree of blood-brain barrier opening based on the number of microbubbles.

[0246] [Table 8]

[0247]

[0248] The results of the analysis of the penetration efficiency of Evans blue under the various opening conditions in Table 8 above, which penetrated the blood-brain barrier and accumulated in the brain, are shown below. Figure 4a As shown in [the image / illustration]. As can be seen in [the image / illustration]. Figure 4a As confirmed by the images and graphs, the increased number of microbubbles leads to a greater degree of opening of the blood-brain barrier, thereby enhancing the penetration efficiency of Evans blue. These results indicate that the increased number of microbubbles resonating with ultrasound leads to increased cavitation, ultimately resulting in increased dye penetration efficiency in brain tissue. Furthermore, H&E staining of brain tissue infiltrated by Evans blue confirmed that Evans blue has no toxicity to brain tissue penetration. Figure 4b ).

[0249] Example 7. Exploration of ultrasound conditions for opening the blood-brain barrier and comparative analysis of Evans blue penetration efficiency.

[0250] Following Example 6, to verify the ultrasound conditions used for opening the blood-brain barrier, the penetration efficiency of Evans blue was quantitatively analyzed based on ultrasound conditions (intensity, frequency, and duty cycle) and ultrasound treatment time. Table 9 shows the comparative experimental design for the degree of blood-brain barrier opening based on ultrasound conditions. The number of injected microbubbles was uniformly 5 × 10⁻⁶. 6 / g.

[0251] [Table 9]

[0252]

[0253] Immediately after intravenous injection of microbubbles, sonication was performed, followed by intravenous administration of 3% (w / w) Evans blue. Twenty-four hours later, brain tissue completely depleted of blood was extracted via perfusion with physiological saline. Quantitative analysis of Evans blue was performed in the same manner as in Example 5 above.

[0254] The results of the analysis of the penetration effect of Evans blue under ultrasound conditions are as follows: Figure 5aAs shown in the figure, the overall opening effect of the blood-brain barrier is enhanced with increasing ultrasound intensity and emission time, and the penetration effect of Evans blue is also increased proportionally. Even in the experimental group receiving the lowest intensity ultrasound treatment (Group 1), it showed a pattern of effective penetration of Evans blue in the tissue at the ultrasound exposure site, and this pattern continued to increase with increasing exposure time. It was also shown that the penetration effect of Evans blue increased with increasing ultrasound intensity and duty cycle.

[0255] Quantitative analysis revealed that increased ultrasound intensity, rather than increased ultrasound emission time, was the factor further enhancing the penetration effect of Evans blue. Furthermore, the increase in duty cycle showed a similar pattern to the increase in Evans blue penetration efficiency with increasing ultrasound exposure time. Figure 5b ).

[0256] However, due to the deaths of some individuals in the aforementioned experimental groups, additional H&E staining was performed to assess the stability of brain tissue under each ultrasound condition in order to determine whether Evans blue penetration of brain tissue was due to a temporary safe opening of the blood-brain barrier or due to brain tissue damage. The results were as follows: Figure 5c As shown, in the experimental groups exposed to high-intensity ultrasound (groups 6 and 7), hemorrhage patterns were observed in a relatively small area compared to the total brain area and the area exposed by ultrasound, while no hemorrhage occurred in the experimental groups exposed to low-intensity ultrasound (groups 1 to 5). Based on these results, the safe ultrasound conditions that do not cause brain tissue damage were confirmed to be the ultrasound conditions in groups 1, 2, and 6.

[0257] Example 8. Analysis of the blood-brain barrier penetration efficiency of acoustic liposomes used to penetrate the blood-brain barrier.

[0258] To analyze the blood-brain barrier penetration efficiency of the acoustic liposomes according to the present invention, the liposomes were labeled with a DiD fluorescent dye displaying near-infrared (NIR) wavelengths and fluorescence analysis was performed using an in vivo imaging system (IVIS). To open the blood-brain barrier, ultrasound was emitted for 60 seconds at 1W, 1% duty cycle, and 1Hz-PRF, the ultrasound conditions obtained in Example 7. The blood-brain barrier was opened in the same manner as described in Examples 6 and 7, and each liposome was immediately injected intravenously once ultrasound emission was complete. Twenty-four hours after liposome injection, organs were extracted and subjected to fluorescence imaging using IVIS. The optimal liposome composition for blood-brain barrier penetration was explored by analyzing the fluorescence images and quantitatively analyzing the fluorescence intensity of each organ.

[0259] In the experiments, all liposomes except for the 004 composition contained sphingomyelin, a representative type of sphingolipid. Sphingomyelin is known to cross the blood-brain barrier, circulate in the blood for extended periods, and has a long half-life. Therefore, to find the optimal composition of liposomes that can effectively cross the blood-brain barrier and accumulate in the brain, the blood-brain barrier penetration efficiency of liposomes was compared and analyzed based on the composition ratio of sphingomyelin. The compositions of the various liposomes used in the experiments are shown in Table 10.

[0260] [Table 10]

[0261]

[0262] Liposomes prepared according to the composition in Table 10 were injected into normal mice and subjected to ultrasound emission under the conditions confirmed as safe for brain tissue in Example 7, namely ultrasound parameters of 1W, 1% DC, and 60s. The blood-brain barrier penetration efficiency and organ distribution results for each liposome were shown in... Figure 6 middle.

[0263] Analysis of organ distribution patterns revealed that all liposomes used in this study exhibited a common trend of accumulating most extensively in the liver, with a relatively high accumulation level also observed in the spleen. These results are attributed to the recognition of liposomes with a size of approximately 100 to 200 nm as foreign bodies in the liver, leading to their excretion. Furthermore, previous studies have also reported a tendency for DOPE to accumulate in the spleen.

[0264] Observation of the brains in the group that did not undergo ultrasound emission showed that liposomes were not detected in all compositions. In other words, the penetration of liposomes into brain tissue appeared to be inhibited by the blood-brain barrier. This is generally because the blood-brain barrier only allows small molecules to pass through actively, and therefore, although sphingomyelin has penetrating capabilities, liposomes with a larger size of approximately 100 to 200 nm cannot cross the blood-brain barrier.

[0265] Conversely, when blood-brain barrier opening was induced using ultrasound and microbubbles, liposomes were detected in the portion exposed to ultrasound, regardless of their composition. No particular trend was observed of increasing delivery efficiency to brain tissue with increasing sphingomyelin content. The liposomes according to the invention exhibited a pattern approximately 2 to 3 times larger than known blood-brain barrier penetration by sphingomyelin. Liposomes with a size of approximately 20 to 50 nm are likely to penetrate the blood-brain barrier, but liposomes reaching 100 nm are less likely to penetrate. To overcome this, when inducing blood-brain barrier opening, the path through which the blood-brain barrier is opened is considered to show a gap greater than 100 nm.

[0266] In addition, to verify the effect of sphingomyelin on the blood-brain barrier penetration of liposomes, a comparative analysis was first performed on liposomes of composition 004 (without sphingomyelin) and liposomes of composition 005 (containing other sphingomyelins). This confirmed that both penetrated the blood-brain barrier at similar levels. Figure 7 ).

[0267] Furthermore, comparative experiments were conducted on the liposomes of compositions 004 and 005 by setting the duty cycle in the ultrasound parameters to 1% or 5%. Other parameters were set to the safe ultrasound parameters confirmed in Example 7. The results, as... Figure 7 As shown, a 5% duty cycle significantly improves the penetration efficiency of both liposomes compared to a 1% duty cycle. Furthermore, the fluorescence intensity increases with increasing duration, indicating improved blood-brain barrier penetration of the liposomes. These results suggest that increasing both the duty cycle and the ultrasound duration can further enhance the blood-brain barrier penetration rate of liposomes.

[0268] Example 9. Comparison of blood-brain barrier penetration effects between acoustic liposomes and Doxil liposomes.

[0269] To compare the delivery effects of the acoustically sensitive liposomes and the non-responsive liposome Doxil for penetrating the blood-brain barrier according to the present invention into the brain, a comparative experiment was conducted by manufacturing liposomes using the 004 composition and the Doxil composition. The experimental methods were performed in the same manner as in the examples described above, and the degree of delivery to brain tissue was quantitatively analyzed by measuring fluorescence intensity using IVIS.

[0270] Experimental results are as follows Figure 8 As shown. Fluorescence was detected at the ultrasound emission site for both the acoustically sensitive liposomes (004 composition liposomes) and the non-responsive liposome Doxil according to the present invention, confirming that both liposomes penetrated the blood-brain barrier due to ultrasound and microbubble cavitation. However, in the case of the acoustically sensitive liposomes according to the present invention, the delivery effect to the brain was found to be almost twice that of the Doxil liposomes. The results indicate that the acoustically sensitive liposomes according to the present invention for penetrating the blood-brain barrier not only have excellent ultrasound responsiveness but also a high penetration effect into brain tissue, enabling more efficient delivery to brain tissue.

[0271] The above description of the present invention is for illustrative purposes only. Those skilled in the art will understand that the technical concept or basic features of the present invention can be readily modified into other specific forms without altering them. Therefore, the above embodiments should be understood as illustrative in all respects and not restrictive.

[0272] [Industrial Applicability]

[0273] This invention relates to acoustic liposomes for penetrating the blood-brain barrier (BBB), which effectively penetrate the BBB upon ultrasound stimulation while exhibiting excellent drug encapsulation efficiency and drug release via ultrasound. Specifically, the acoustic liposomes according to the invention can circulate in vivo for extended periods, thus exhibiting excellent BBB penetration efficiency and high affinity for brain tumor cells, resulting in outstanding delivery to tumor sites. Therefore, the acoustic liposomes according to the invention can be used as drug delivery carriers for delivering therapeutic agents for brain diseases to the brain. In particular, the inventors have determined optimal cavitation conditions for stably opening the BBB to further enhance the drug delivery effect of the acoustic liposomes. It is anticipated that when these cavitation conditions are combined with the acoustic liposomes according to the invention for penetrating the BBB, excellent therapeutic effects can be achieved in various brain diseases.

Claims

1. A method for manufacturing a brain-targeting drug delivery vehicle capable of penetrating the blood-brain barrier, the brain-targeting drug delivery vehicle comprising acoustic liposomes, the method comprising the following steps: (S1) Dissolve DSPC, DSPE-mPEG2000, DOPE, cholesterol, lyso-PC and polysorbate in the first organic solvent; (S2) Evaporate the organic solvent to produce a liposome membrane; and (S3) Hydrate the liposome membrane with an aqueous solution. The first organic solvent is at least one selected from the group consisting of: dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and diethyl ether. The acoustic liposomes described herein penetrate the blood-brain barrier opened by ultrasonic cavitation, and The molar percentages of the DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC contained therein are 8~12 : 3~6 : 50~66 : 10~32 : 5~11.

2. The method of claim 1, wherein the sonosensitive liposome is at least one selected from the group consisting of: (a) Particle size of 100 to 200 nm; and (b) The proportion of the drug encapsulated in the liposomes to the total added drug is 50-100%.

3. The method according to claim 1, wherein the drug is a treatment agent for brain diseases. The brain disease mentioned herein is selected from at least one of the following groups: brain tumor, brain infection caused by bacteria or virus, Parkinson's disease, encephalitis, stroke, paralysis, Alzheimer's disease, Lujarig's disease, Huntington's disease, Pick's disease, Kreutzfeldt-Jacob's disease, epilepsy, and brain metabolic disorders. The brain disease treatment agent mentioned herein is at least one selected from the group consisting of: vincristine, vinblastine, vinflunidine, vindesine, vinorelbine, temozolomide, carmustine, lomustine, cabazitaxel, docetaxel, larotaxel, oxalitaxel, paclitaxel, testacrol, ixaspirin, procarbazine, rituximab, tocilizumab, carboplatin, erlotinib, irinotecan, enzartolin, vorinostatin, doxorubicin, cisplatin, 5-fluorouracil, tamoxifen. Topotecan, belotetane, imatinib, fluorouracil, gemcitabine, leuprorelin, flutamide, zoledronic acid, methotrexate, camptothecin, hydroxyurea, streptozocin, penoxuridine, retinic acid, nitrogen mustard, chlorambucil, busulfan, doxyfluridine, mitomycin, prednisone, everolimus, mitoxantrone, levodopa, carbidopa, entacapone, tocapone, dopamine agonists, donepezil, galantamine, rivastigmine, memantine, anticholinergics, and amantadine.

4. The method according to claim 1, wherein the sonosensitive liposomes are hydrated with ammonium sulfate, ammonium citrate or TEA-SOS.

5. The method of claim 1, wherein the brain-targeting drug delivery vehicle is administered sequentially or simultaneously with ultrasound treatment, and The ultrasonic treatment is selected from at least one of the following: (a) The frequency of the ultrasound is from 20 kHz to 3 MHz; and (b) Duty cycle of 0.5% to 20%.

6. The method of claim 5, further comprising applying microbubbles, wherein the ultrasonic treatment is performed sequentially or simultaneously with the application of the microbubbles.

Images