Sound-sensitive liposomes and their preparation method

Abstract

The present invention relates to sonosensitive liposomes and a method for preparing the same. The sonosensitive liposomes according to the present invention improve the delivery efficiency of encapsulated drugs, and are expected to exhibit various functions in the fields of cancer treatment and drug delivery carriers by improving drug delivery effects through targeted cancer cells when used in combination with ultrasonic treatment.

Description

Technical Field

[0001] The present invention relates to sound-sensitive liposomes and a method for preparing the same.

[0002] This application claims the priority and benefits of Korean Patent Application Nos. 10-2020-0052356 and 10-2021-0042529, filed with the Korean Intellectual Property Office (KIPO) on April 29, 2020, and April 1, 2021, respectively, and incorporates all the content disclosed in the specifications and drawings of these applications into this application. Background Art

[0003] Cancer is one of the incurable diseases that humanity must address, and a huge amount of investment is being made worldwide to develop drugs for treating cancer. In Korea, as the leading cause of death, more than 100,000 people are diagnosed every year, and more than 60,000 people die.

[0004] Currently, cancer treatment methods used clinically include chemotherapy, radiotherapy, targeted therapy, methods of removing lesions through surgery, etc. Representative examples of chemotherapeutic agents used for anti-cancer treatment to date include doxorubicin or adriamycin, cisplatin, paclitaxel, 5-fluorouracil, etc., and they have been widely used in cancer treatment chemotherapy. However, these methods have limitations and cannot completely cure the disease, but they cause serious side effects and pain to patients. Therefore, there is a need to develop cancer treatment technologies that can minimize side effects.

[0005] Meanwhile, liposomes are vesicles composed of phospholipids and their derivatives. Phospholipids and their derivatives spontaneously form vesicles when dispersed in water, which are characterized by a lipid bilayer surrounding the cell nucleus composed of an aqueous phase. In the fields of medicine, pharmacy, and biochemistry, various liposomes have been used as carriers for therapeutic agents such as drugs, enzymes, gene sequences, etc.

[0006] Therefore, the present inventors have developed a new method for treating cancer by integrating ultrasonic technology with liposome technology, which is still unknown in the research and development fields related to cancer such as cancer diagnosis and treatment. Summary of the Invention

[0007] [Technical Problem]

[0008] The present inventors have developed sound-sensitive liposomes having excellent drug release rates and excellent stability.

[0009] More specifically, the present inventors have found that when ultrasound is applied to drug-containing liposomes through the integration of ultrasonic technology, compared with when drug-containing liposomes are not treated with ultrasound, the drug-containing liposomes have a high drug release rate and excellent anti-tumor effects, and also exhibit excellent blood stability. Therefore, the present invention has been completed based on the above facts.

[0010] Accordingly, one aspect of the present invention is to provide a sonosensitive liposomal formulation, which comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol 2000) (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and lysophosphatidylcholine (lyso-PC).

[0011] Another aspect of the present invention is to provide a sonosensitive liposomal formulation, which comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol 2000) (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and lysophosphatidylcholine (lyso-PC).

[0012] Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the sonosensitive liposomal formulation.

[0013] Another aspect of the present invention is to provide a composition for drug delivery, which comprises a liposome as an active ingredient, and a drug is encapsulated in the liposome, wherein the liposome comprises DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine (lyso-PC); or comprises DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lysophosphatidylcholine (lyso-PC).

[0014] Another aspect of the present invention is to provide a method for preparing sonosensitive liposomes.

[0015] However, the technical objects of the present invention are not limited thereto. By describing the exemplary embodiments of the present invention in detail, those of ordinary skill in the art will be more clear about other objects of the present invention not described herein.

[0016] [Technical Solution]

[0017] According to one aspect of the present invention, there is provided a sonosensitive liposomal formulation, which comprises:

[0018] 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol 2000) (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and lysophosphatidylcholine (lyso-PC); or

[0019] 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol 2000) (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and lysophosphatidylcholine.

[0020] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, which comprises a sonosensitizing liposomal preparation.

[0021] According to still another aspect of the present invention, there is provided a composition for drug delivery, which comprises a sonosensitizing liposome as an active ingredient, and a drug is encapsulated in the sonosensitizing liposome, wherein the liposome comprises DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine; or comprises DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lysophosphatidylcholine.

[0022] According to another aspect of the present invention, there is provided a method for preparing a sonosensitizing liposome, which comprises one of the following preparation processes 1 to 3:

[0023] [Preparation Process 1]

[0024] (a) Dissolve DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine in a first organic solvent;

[0025] (b) Evaporate the organic solvent to prepare a liposomal membrane;

[0026] (c) Hydrate the liposomal membrane in an aqueous solution and stir the liposomal membrane solution; and

[0027] (d) Extrude the liposomal membrane solution through an extruder.

[0028] [Preparation Process 2]

[0029] (a) Dissolve DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine in ethanol;

[0030] (b) Hydrate the ethanol solution prepared in step (a) in an aqueous solution and stir the ethanol solution; and

[0031] (c) Extrude the ethanol solution through an extruder.

[0032] [Preparation Process 3]

[0033] (a) Dissolve DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine in a first organic solvent;

[0034] (b) Mix the first organic solvent solution prepared in step (a) and the aqueous solution while using a microfluidic system

[0035] change the flow rate of the channels of the system; and

[0036] (c) Remove the organic solvent.

[0037] According to another aspect of the present invention, there is provided a method for treating cancer, which includes: administering an effective amount of a sonosensitizing liposomal preparation to a subject; and sonicating the sonosensitizing liposomal preparation.

[0038] According to another aspect of the present invention, there is provided the use of a sonosensitizing liposomal preparation in cancer treatment.

[0039] According to another aspect of the present invention, there is provided the use of a sonosensitizing liposomal preparation in the preparation of a cancer therapeutic agent.

[0040] According to another aspect of the present invention, there is provided a method for delivering a drug, which includes: administering an effective amount of a liposome encapsulating the drug to a subject, wherein the liposome includes DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine.

[0041] According to another aspect of the present invention, there is provided the use of a liposome encapsulating a drug for drug delivery, wherein the liposome includes DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine.

[0042] According to another aspect of the present invention, there is provided the use of a liposome encapsulating a drug in the preparation of a drug delivery carrier, wherein the liposome includes DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine.

[0043] According to an exemplary embodiment of the present invention, it may include DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lysophosphatidylcholine in a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20, but the present invention is not limited thereto.

[0044] According to another exemplary embodiment of the present invention, DSPC may be included in a dry weight of 0.1 to 50% based on the total weight of the liposomal preparation, DSPE-mPEG2000 may be included in a dry weight of 3 to 50% based on the total weight of the liposomal preparation, DOPE may be included in a dry weight of 1 to 80% based on the total weight of the liposomal preparation, cholesterol may be included in a dry weight of 0.05 to 40% based on the total weight of the liposomal preparation, and lysophosphatidylcholine may be included in a dry weight of 0.5 to 10% based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0045] According to yet another exemplary embodiment of the present invention, the liposome membrane of the sonosensitive liposome preparation may be composed of DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lysophosphatidylcholine, but the present invention is not limited thereto.

[0046] According to yet another exemplary embodiment of the present invention, a drug may be encapsulated in the sonosensitive liposome preparation, but the present invention is not limited thereto.

[0047] According to yet another exemplary embodiment of the present invention, the drug may be an anticancer drug and may include, for example, one or more selected from the group consisting of doxorubicin, paclitaxel, docetaxel, cisplatin, Gleevec, 5-fluorouracil (5-FU), tamoxifen, carboplatin, topotecan, belotecan, imatinib, irinotecan, fluphenazine, vinorelbine, gemcitabine, leuprorelin, flutamide, zoledronate, methotrexate, camptothecin, vincristine, hydroxyurea, streptozotocin, valrubicin, retinoic acid, nitrogen mustard, chlorambucil, busulfan, doxifluridine, vinblastine, mitomycin, prednisone, everolimus, and mitoxantrone, but the present invention is not limited thereto.

[0048] According to yet another exemplary embodiment of the present invention, the sonosensitive liposome preparation may have ensured stability, and the stability may be blood stability, but the present invention is not limited thereto.

[0049] According to yet another exemplary embodiment of the present invention, the sonosensitive liposome preparation may have one or more of the following characteristics, but the present invention is not limited thereto:

[0050] i. The particle size is 100 nm to 200 nm; and

[0051] ii. The drug encapsulation efficiency is 50% to 100%.

[0052] According to yet another exemplary embodiment of the present invention, the sonosensitive liposome preparation or a pharmaceutical composition comprising the sonosensitive liposome preparation may be administered sequentially or simultaneously with sonication, but the present invention is not limited thereto.

[0053] According to yet another exemplary embodiment of the present invention, cancer may include one or more selected from the group consisting of: breast cancer, colon cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colorectal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumors, primary CNS lymphoma, spinal cord tumors, brainstem glioma, and pituitary adenoma, but the present invention is not limited thereto.

[0054] According to yet another exemplary embodiment of the present invention, in step (a), cholesterol may be further added and dissolved therein, but the present invention is not limited thereto.

[0055] According to yet another exemplary embodiment of the present invention, the organic solvent may include one or more selected from the group consisting of: dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether, but the present invention is not limited thereto.

[0056] [Beneficial Effects]

[0057] The inventors have found that the sonosensitive liposomes according to the present invention have high encapsulation efficiency, excellent drug stability and liposome preparation stability, and show excellent effects of increasing the drug release rate by sonication, as well as cancer cell lethal effect and cancer cell penetration effect, compared with existing commercial drugs. Therefore, the sonosensitive liposomes according to the present invention improve the delivery efficiency of the encapsulated drug and have improved drug delivery effects when used in combination with sonication to target cancer cells. Therefore, the sonosensitive liposomes according to the present invention are expected to show various functions in the fields of cancer treatment and drug delivery carriers. Brief Description of the Drawings

[0058] Figure 1 is a graph showing the doxorubicin release rate of sonosensitive liposomes according to the ratio of 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (MSPC) according to an exemplary embodiment of the present invention.

[0059] Figure 2 is a graph showing the doxorubicin release rate of sonosensitive liposomes according to the change in cholesterol ratio according to an exemplary embodiment of the present invention.

[0060] Figure 3It is a graph showing the doxorubicin release rate of each liposome containing DOPC and DOPE respectively by ultrasonic sensitization according to an exemplary embodiment of the present invention.

[0061] Figure 4 It is a graph showing the doxorubicin release rate of each liposome by ultrasonic sensitization according to the DSPC ratio according to an exemplary embodiment of the present invention.

[0062] Figure 5 It is a graph showing the pretreatment process for evaluating the 75% serum stability of liposomes according to an exemplary embodiment of the present invention.

[0063] Figure 6 It is a graph showing the average doxorubicin release rate according to the MSPC ratio according to an exemplary embodiment of the present invention, which shows the analysis of the doxorubicin release rate according to the MSPC ratio in the 75% serum stability evaluation test.

[0064] Figure 7 It is a graph showing the comparative evaluation results of the doxorubicin release rate (left) and the particle size change trend analysis (right) over time and in the presence / absence of ultrasonic irradiation according to an exemplary embodiment of the present invention.

[0065] Figure 8 It is a graph showing the drug release rate of sonosensitive liposomes containing various types of hemolytic-PC (14:0, 16:0, 17:0, 17:1, 18:0, 18:1) according to an exemplary embodiment of the present invention.

[0066] Figure 9 It is a graph showing the cancer cell lethal effect of sonosensitive liposomes in the presence / absence of irradiation and the control Caelyx with ultrasound according to an exemplary embodiment of the present invention.

[0067] Figure 10 It is a graph showing the doxorubicin cell penetration effect of sonosensitive liposome irradiation and the control Caelyx with ultrasound according to an exemplary embodiment of the present invention.

[0068] Figure 11 It is a graph showing the doxorubicin plasma concentration changing over time according to the MSPC ratio according to an exemplary embodiment of the present invention, which shows the determination of the doxorubicin plasma concentration according to the MSPC ratio in the blood pK evaluation test. Detailed Description

[0069] The present invention provides a sound-sensitive liposomal preparation, which comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol 2000) (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and lysophosphatidylcholine (lyso-PC).

[0070] The preparation of the present invention may further comprise cholesterol.

[0071] The term "DSPC" used in the present invention is an abbreviation of 1,2-distearoyl-sn-glycero-3-phosphocholine, and refers to a phospholipid composed of two stearic acid residues linked to a phosphatidylcholine head group.

[0072] The term "DSPE-mPEG2000" used in the present invention is an abbreviation of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol 2000), and refers to a polyethylene glycolated derivative of 1,2-distearoyl-sn-glycero-3-PE (DSPE).

[0073] The term "DOPE" used in the present invention is an abbreviation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and it is known that it forms heterogeneous liposomes with DOTAP, where DOTAP is used as a delivery carrier for therapeutic agents.

[0074] The term "cholesterol" used in the present invention refers to one of the steroid compounds. Cholesterol includes cholesterol derivatives. Cholesterol derivatives can be, for example, sitosterol, ergosterol, stigmasterol, 4,22-stigmast-3-one, stigmasterol acetate, lanosterol, cycloeucalenol, or a combination thereof. Cholesterol is located in the lipid bilayer, and its amount is adjusted to decrease or increase permeability. In this case, cholesterol can be used regardless of its own ratio in the liposomal preparation.

[0075] The term "lyso-PC" used in the present invention is an abbreviation of lysophosphatidylcholine, which is a representative term for lipids composed of acyl chains derived from phosphocholine (which consists of choline as the head group), and includes various types of lyso-PC.

[0076] In the present invention, lyso-PC can be represented by the following formula 1, but the present invention is not limited thereto.

[0077] [Formula 1]

[0078]

[0079] (wherein R1 and / or R2 is C6 to C 26Acyl group, C6 to C 26 Alkyl group, C6 to C 26 Alkenyl group, C6 to C 26 Alkynyl group, substituted or unsubstituted C6 to C 26 Cycloalkyl, substituted or unsubstituted C6 to C 26 Aryl, substituted or unsubstituted C7 to C 26 Aralkyl group, or H).

[0080] In the present invention, the acyl group may be an aromatic carboxylic acid residue, a saturated fatty acid residue or an unsaturated fatty acid residue, but the present invention is not limited thereto.

[0081] In the present invention, the lysophosphatidylcholine (LPC) represented by Formula 1 may be more particularly represented by the following Formula 2 or 3, but the present invention is not limited thereto.

[0082] In the present invention, the lysophosphatidylcholine (LPC) may be 1-lysophosphatidylcholine (LPC) or 2-LPC represented by the following Formula 2 or 3, but the present invention is not limited thereto.

[0083] [Formula 2]

[0084]

[0085] [Formula 3]

[0086]

[0087] (wherein R is C6 to C 26 Alkyl group, C6 to C 26 Alkenyl group, C6 to C 26 Alkynyl group, substituted or unsubstituted C6 to C 26 Cycloalkyl, substituted or unsubstituted C6 to C 26 Aryl group, substituted or unsubstituted C7 to C 26 Alkyl group, or H).

[0088] In the present invention, lysophosphatidylcholine (lyso-PC) may include one or more selected from the group consisting of: lysophosphatidylcholine (6:0), lysophosphatidylcholine (7:0), lysophosphatidylcholine (8:0), lysophosphatidylcholine (9:0), lysophosphatidylcholine (10:0), lysophosphatidylcholine (11:0), lysophosphatidylcholine (12:0), lysophosphatidylcholine (13:0), lysophosphatidylcholine (14:0), lysophosphatidylcholine (15:0), lysophosphatidylcholine (16:0), 2-lysophosphatidylcholine (16:0), lysophosphatidylcholine (17:0), lysophosphatidylcholine (17:1), lysophosphatidylcholine (18:0), lysophosphatidylcholine (18:1), lysophosphatidylcholine (18:2), 2-lysophosphatidylcholine (18:0), 2-lysophosphatidylcholine (18:1), lysophosphatidylcholine (19:0), lysophosphatidylcholine (20:1), lysophosphatidylcholine (20:4), lysophosphatidylcholine (22:0), lysophosphatidylcholine (22:5), lysophosphatidylcholine (24:0), lysophosphatidylcholine (26:0), and lysophosphatidylcholine (20:5), but the present invention is not limited thereto.

[0089] DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine may be included at a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 20, but the present invention is not limited thereto.

[0090] According to another exemplary embodiment of the present invention, DSPC may be included at a dry weight of 0.1 to 50% based on the total weight of the liposomal formulation, DSPE-mPEG2000 may be included at a dry weight of 3 to 50% based on the total weight of the liposomal formulation, DOPE may be included at a dry weight of 1 to 80% based on the total weight of the liposomal formulation, and lysophosphatidylcholine may be included at a dry weight of 0.5 to 10% based on the total weight of the liposomal formulation, but the present invention is not limited thereto.

[0091] In addition, in the present invention, DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lysophosphatidylcholine may be included in the following molar ratios (mol%): a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20, a molar ratio (mol%) of 1 to 40:1 to 9:35 to 80:0.1 to 40:1 to 10, a molar ratio (mol%) of 10 to 40:3 to 7:40 to 65:0.1 to 45:0.1 to 10, a molar ratio (mol%) of 0.1 to 20:1 to 8:63 to 67:0.1 to 30:3 to 10, a molar ratio (mol%) of 5 to 15:2 to 7:64 to 67:10 to 30:3 to 10, a molar ratio (mol%) of 5 to 15:3 to 6:64 to 66:15 to 30:5 to 10, a molar ratio (mol%) of 0.1 to 20:1 to 8:53 to 57:0.1 to 30:5 to 10, a molar ratio (mol%) of 5 to 15:2 to 7:54 to 57:3 to 20:5 to 10, or a molar ratio (mol%) of 5 to 15:3 to 6:54 to 56:5 to 10:5 to 10, but the present invention is not limited thereto.

[0092] In addition, in the present invention, DSPC may be included in a molar ratio (mol%) of 1 to 50, 5 to 50, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 7 to 12, 25 to 35, 27 to 32, or 35 to 45 based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0093] In addition, in the present invention, DSPE-mPEG2000 may be included in a molar ratio (mol%) of 1 to 10, 2 to 9, 3 to 8, 3 to 7, or 4 to 6 based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0094] In addition, in the present invention, DOPE may be included in a molar ratio (mol%) of 5 to 80, 5 to 70, 10 to 70, 20 to 70, 30 to 70, 40 to 70, or 40 to 65 based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0095] In addition, in the present invention, cholesterol can be included at a molar ratio (mol%) of 0.1 to 50, 0.1 to 45, 1 to 45, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 10 to 40, 10 to 30, 10 to 25, 10 to 20, 20 to 40, 30 to 40, 5 to 10, or 40 to 50 based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0096] In addition, in the present invention, lyso-PC can be included at a molar ratio (mol%) of 0.1 to 20, 0.1 to 15, 0.1 to 10, 1 to 20, 3 to 20, 3 to 15, 3 to 10, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 7 to 20, 7 to 15, 7 to 12, 8 to 12, 9 to 11, 4 to 6, or 6 to 8 based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0097] In addition, in the present invention, DSPC can be included at 0.1 to 50% dry weight based on the total weight of the liposomal preparation, DSPE-mPEG2000 can be included at 3 to 50% dry weight based on the total weight of the liposomal preparation, DOPE can be included at 1 to 80% dry weight based on the total weight of the liposomal preparation, cholesterol can be included at 0.05 to 40% dry weight based on the total weight of the liposomal preparation, and lyso-PC can be included at 0.5 to 10% dry weight based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0098] In the present invention, DSPC can be included at 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 based on the total weight of the liposomal formulation, but the present invention is not limited thereto.

[0099] In the present invention, DSPE-mPEG2000 can be included at 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 based on the total weight of the liposomal formulation, but the present invention is not limited thereto.

[0100] In the present invention, cholesterol can be included at 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% dry weight based on the total weight of the liposomal formulation, but the present invention is not limited thereto.

[0101] In the present invention, DOPE can be included at 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 based on the total weight of the liposomal formulation, but the present invention is not limited thereto.

[0102] In the present invention, hemolytic-PC may be included at 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, 2 to 4% dry weight, 2.5 to 7.5% dry weight, 2.5 to 7% dry weight, 2.5 to 6.5% dry weight, 3 to 8% dry weight, 3 to 7% dry weight, 3 to 6% dry weight, 3 to 5% dry weight, 3 to 4% dry weight, 5 to 10% dry weight, 5 to 9% dry weight, 5 to 8% dry weight, 5 to 7% dry weight, 5 to 6% dry weight, or 5 to 5.5% dry weight based on the total weight of the liposomal preparation, but the present invention is not limited thereto.

[0103] In the present invention, the liposomal membrane of the sonosensitive liposomal preparation may be composed of DSPC, DSPE-mPEG2000, DOPE, cholesterol, and hemolytic-PC, but the present invention is not limited thereto.

[0104] The liposomes of the present invention are formed from amphiphilic compounds including phospholipids. Such amphiphilic compounds are typically arranged at the interface between an aqueous medium and an inherently immiscible organic solvent to stabilize emulsified solvent droplets. Amphiphilic compounds include compounds having molecules that contain a hydrophilic polar head portion (e.g., a polar or ionic group) capable of reacting with the aqueous medium and a hydrophobic organic tail portion (e.g., a hydrocarbon chain) capable of reacting with, for example, the organic solvent. Amphiphilic compounds are compounds that can stabilize mixtures of materials that cannot otherwise be mixed, such mixtures as a mixture of two immiscible liquids (e.g., water and oil), a mixture of a liquid and a gas (e.g., gas microbubbles in water), or a mixture of a liquid and insoluble particles (e.g., metal nanoparticles in water).

[0105] The term "ultrasound" as used in the present invention generally refers to sound waves having a frequency greater than 16 Hz to 20 kHz (corresponding to the frequency of sound waves audible to humans). High-intensity focused ultrasound can have instantaneous thermal effects (65 - 100 °C), cavitation effects, mechanical effects, and sonochemical effects according to the energy and number of vibrations by introducing focused ultrasound that provides continuous and high-intensity ultrasound energy to the focus. Ultrasound is harmless to the human body when passing through human tissues, but the high-intensity ultrasound forming the focus generates a sufficient amount of energy to cause coagulative necrosis and thermal ablation effects regardless of the tissue type.

[0106] According to the present invention, ultrasound refers to sound waves having a frequency greater than 16 Hz to 20 kHz (corresponding to the range of audible frequencies). The ultrasound can be high-intensity focused ultrasound (HIFU), high-intensity non-focused ultrasound, or a combination thereof, but the present invention is not limited thereto. HIFU refers to ultrasound that focuses high-intensity ultrasound energy at a point to form a focal point. HIFU can be ultrasound-guided high-intensity focused ultrasound (ultrasound-guided HIFU) and magnetic resonance imaging-guided high-density focused ultrasound (MRI-guided HIFU), depending on what high-intensity focused ultrasound treatment is performed when viewing any particular image. The frequency of the ultrasound can be, for example, in the following ranges: 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 to 2.0 MHz, 300 kHz to 2.0 MHz, 350 kHz to 2.0 MHz, 400 kHz to 2.0 MHz, 450 kHz to 2.0 MHz, 500 kHz to 2.0 MHz, 550 kHz to 2.0 MHz, 600 kHz to 2.0 MHz, 650 kHz to 2.0 MHz, 700 kHz to 2.0 MHz, 750 kHz to 2.0 MHz, 800 kHz to 2.0 MHz, 850 kHz to 2.0 MHz, 900 kHz to 2.0 MHz, 950 kHz to 2.0 MHz, 600 kHz to 1.5 MHz, 650 kHz to 1.5 MHz, 700 kHz to 1.5 MHz, 750 kHz to 1.5 MHz, 800 kHz to 1.5 MHz, 850 kHz to 1.5 MHz, 900 kHz to 1.5 MHz, 950 kHz to 1.5 MHz, 1 MHz to 1.5 MHz, 600 kHz to 1.3 MHz, 650 kHz to 1.3 MHz, 700 kHz to 1.3 MHz, 750 kHz to 1.3 MHz, 800 kHz to 1.3 MHz, 850 kHz to 1.3 MHz, 900 kHz to 1.3 MHz, 950 kHz to 1.3 MHz, 600 kHz to 1.1 MHz, 650 kHz to 1.1 MHz, 700 kHz to 1.1 MHz, 750 kHz to 1.1 MHz, 800 kHz to 1.1 MHz, 850 kHz to 1.1 MHz, 900 kHz to 1.1 MHz, or 950 kHz to 1.1 MHz, but the present invention is not limited thereto.

[0107] The liposome is a sonosensitive liposome. The term "sonosensitive liposome" refers to a liposome whose permeability increases when the liposome is exposed to ultrasound. Therefore, when the liposome is exposed to ultrasound, the drug contained in the liposome may be released.

[0108] Liposomes can have ensured stability. The term "stability" refers to the situation where the drug encapsulated in the blood liposomes is not released when the liposomes are not exposed to ultrasound. According to a specific embodiment, when the release rate of the drug encapsulated in the liposomes is measured every 20 minutes for 60 minutes under vortex conditions (60 minutes) at room temperature (20 to 25 °C), the liposomes have a drug release rate of up to 30% or lower.

[0109] Liposomes can have the following particle sizes (e.g., diameters): 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 approximately 150 nm. According to a specific embodiment, the liposomes can have a particle size of 100 nm to 200 nm. Here, the particle size measurement method described in Example 1 of this specification is used, but the present invention is not limited thereto. In this case, other methods known in the art can be used to measure the particle size, and it can be converted to an equivalent value level.

[0110] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises a sonosensitive liposome preparation.

[0111] In addition, the present invention provides a composition for drug delivery, which comprises sonosensitive liposomes encapsulating a drug as an active ingredient, wherein the liposomes comprise DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine; or comprise DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lysophosphatidylcholine.

[0112] The term "drug" used in the present invention refers to any compound having the desired biological activity. The desired biological activity includes activities for diagnosing, curing, alleviating, treating, or preventing diseases in humans or other animals.

[0113] In the present invention, the drug may be an anti-cancer drug and may include, for example, one or more selected from the group consisting of doxorubicin, paclitaxel, docetaxel, cisplatin, Gleevec, 5-fluorouracil (5-FU), tamoxifen, carboplatin, topotecan, belotecan, imatinib, irinotecan, fluphenazine, vinorelbine, gemcitabine, leuprorelin, flutamide, zoledronate, methotrexate, camptothecin, vincristine, hydroxyurea, streptozotocin, valrubicin, retinoic acid, nitrogen mustard, chlorambucil, busulfan, doxifluridine, vinblastine, mitomycin, prednisone, everolimus, and mitoxantrone, but the present invention is not limited thereto.

[0114] According to the present invention, the drug and the sonosensitizing liposomes may preferably be mixed at a mass ratio (w / w%) of 1:2 to 1:20, more preferably at a mass ratio (w / w%) of 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 1:10.5, 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, but the mixing ratio of the drug to the sonosensitizing liposomes is not limited as long as the drug can be effectively encapsulated in the liposomes.

[0115] According to the present invention, the term "encapsulation efficiency" may be used interchangeably with the encapsulation efficiency. According to the present invention, the sonosensitizing liposomes encapsulating the drug may have an anti-cancer drug encapsulation efficiency of 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 the present invention is not limited thereto.

[0116] The encapsulation efficiency refers to the encapsulation efficiency relative to the amount of the drug added, but the present invention is not limited thereto. Here, the method for measuring the drug encapsulation efficiency is carried out by the method described in Example 1 of this specification, but the present invention is not limited thereto. In this case, other methods known in the art may be used to measure the drug encapsulation efficiency and converted to an equivalent value level.

[0117] According to the present invention, the drug-encapsulated sonosensitive liposomes may have a drug release rate of 10% to 100%, 50% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, or 75% to 100%, but the present invention is not limited thereto. The drug release rate may refer to the release rate relative to the amount of the added drug, but the present invention is not limited thereto. Here, the method for measuring the release rate is carried out by the method described in Example 2 of this specification, but the present invention is not limited thereto. In this case, other methods known in the art may be used to measure the release rate and it may be converted to an equivalent value level.

[0118] According to the present invention, when the sonosensitive liposomes are not ultrasonically treated, the drug release rate of the drug-encapsulated sonosensitive liposomes in the blood may be 0.1% to 100%, 0.1% to 80%, 0.1% to 60%, 0.1% to 40%, 0.1% to 30%, 0.1% to 20%, or 30% or less, but the present invention is not limited thereto.

[0119] In the present invention, the ultrasound-sensitive liposome preparation or the pharmaceutical composition comprising the ultrasound-sensitive liposome preparation may be administered sequentially or simultaneously with ultrasonic treatment, but the present invention is not limited thereto.

[0120] The term "cancer" as used in the present invention generally refers to all types of diseases caused in cells that have: the proliferative characteristics of cell division and growth while ignoring normal growth limitations, the invasive characteristics of cells penetrating into surrounding tissues, and the metastatic characteristics of cells spreading to other parts of the body.

[0121] According to the present invention, the type of cancer is not particularly limited as long as it is a malignant tumor known in the art. For example, cancer may include one or more selected from the group consisting of: breast cancer, colon cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, uveal melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colorectal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumors, primary CNS lymphoma, spinal cord tumors, brainstem glioma, and pituitary adenoma.

[0122] According to the present invention, the term "prevention" refers to all types of actions that inhibit or delay the onset of cancer by administering the composition according to the present invention.

[0123] According to the present invention, the term "treatment" refers to all types of actions that alleviate or are beneficial to cancer and its symptoms by administering the composition according to the present invention.

[0124] In the present invention, the term "pharmaceutical composition" refers to a composition prepared for preventing or treating cancer, and can be formulated into various forms and used by each conventional method. For example, the pharmaceutical composition can be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be formulated into forms such as topical skin preparations (e.g., creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes, poultices, etc.), suppositories, and sterile injection solutions for treatment.

[0125] The pharmaceutical composition according to the present invention may further include suitable carriers, excipients or diluents commonly used in preparing pharmaceutical compositions. In this case, the carriers, excipients and diluents that can be included in the composition may include lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil.

[0126] When formulating, the composition can be prepared using common diluents or excipients (such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.).

[0127] Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing at least one excipient (e.g., starch, calcium carbonate, sucrose or lactose, gelatin, etc.) with the extract. In addition, lubricants such as magnesium stearate, talc, etc. are used in addition to simple excipients. Liquid preparations for oral administration may include suspensions, oral liquids, emulsions, syrups, etc. In addition to common simple diluents (such as water, liquid paraffin, etc.), liquid preparations may also include various excipients such as wetting agents, sweeteners, flavors, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), injectable esters (such as ethyl oleate), etc. can be used as non-aqueous solvents and suspending agents. Witepsol, Macrogol, Tween 61, cocoa butter, lauric acid esters, glycerogelatin, etc. can be used as the matrix of suppositories.

[0128] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or by topical application) according to the desired method, and the dosage can depend on the condition and weight of the patient, the severity of the disease, the type of drug, the route of administration, and the duration of administration, but can be appropriately selected by those skilled in the art.

[0129] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. According to the present invention, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective dose can be determined according to factors such as the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the duration of administration, the route of administration and the secretion rate, the treatment cycle, factors including co-administered drugs, and other factors well known in the medical field. The pharmaceutical composition according to the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents. In this case, the pharmaceutical composition can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. Considering all the above factors, it is important to administer a certain amount of the pharmaceutical composition that can achieve the maximum effect with the minimum amount. In this case, the amount of the pharmaceutical composition can be easily determined by those skilled in the art. The dosage can be administered once a day or in equally divided doses.

[0130] The pharmaceutical composition for preventing or treating cancer according to the present invention can be used in combination with liposomes contained in the composition, and in addition to sonosensitizing liposomes, the composition can further include one or more selected from the group consisting of: anticancer drugs, imaging contrast agents, antibiotics, anti-inflammatory agents, proteins, cytokines, peptides, and antibodies.

[0131] According to the present invention, the term "administration" refers to the process of providing a predetermined amount of the composition of the present invention to a subject using any suitable method.

[0132] According to the present invention, the term "subject" refers to a target in need of treatment for a disease to which the composition of the present invention can be administered. More specifically, the subject refers to a mammal, such as a human or non-human primate, mouse, dog, cat, horse, cow, etc.

[0133] In addition, the present invention provides a method for preparing sonosensitizing liposomes, which includes one of the following preparation processes 1 to 3:

[0134] [Preparation Process 1]

[0135] (a) Dissolve DSPC, DSPE-mPEG2000, DOPE, and lysophosphatidylcholine in a first organic solvent;

[0136] (b) Evaporate the organic solvent to prepare a liposome membrane;

[0137] (c) Hydrate the liposome membrane in an aqueous solution and stir the liposome membrane solution; and

[0138] (d) Extrude the liposome membrane solution through an extruder.

[0139] [Preparation Process 2]

[0140] (a) Dissolve DSPC, DSPE-mPEG2000, DOPE and lyso-PC in ethanol;

[0141] (b) Hydrate the ethanol solution prepared in step (a) in an aqueous solution and stir the ethanol solution; and

[0142] (c) Extrude the ethanol solution through an extruder.

[0143] [Preparation Process 3]

[0144] (a) Dissolve DSPC, DSPE-mPEG2000, DOPE and lyso-PC in a first organic solvent;

[0145] (b) Mix the first organic solvent solution prepared in step (a) and an aqueous solution, while changing the flow rate thereof using the channels of a microfluidic system

[0146] system; and

[0147] (c) Remove the organic solvent.

[0148] In the present invention, Preparation Process 2 may further include evaporating ethanol after step (b), but the present invention is not limited thereto.

[0149] In the present invention, the aqueous solution may be an aqueous solution of ammonium sulfate, but the present invention is not limited thereto.

[0150] In the present invention, removing the organic solvent may include evaporating the organic solvent, but the present invention is not limited thereto.

[0151] In the present invention, the microfluidic system uses a known high-pressure homogenization method using a high-pressure emulsification device, but the present invention is not limited thereto. For information on the microfluidic system, reference can be made to Whitesides, G.M. (2006). The origins and the future of microfluidics. Nature, 442(7101), 368 - 373.

[0152] In the present invention, the first organic solvent may include one or more selected from the group consisting of: dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol and ether, but the present invention is not limited thereto.

[0153] The liposomal preparation prepared by the method of the present invention can be used as an injection and can be used for percutaneous and intranasal drug delivery to the lungs. The techniques required for such a preparation, as well as pharmaceutically suitable carriers, additives, etc. are well known to those of ordinary skill in the pharmaceutical art and reference can be made to Remington’s Pharmaceutical Sciences (19th edition, 1995).

[0154] In addition, the temperature required for extruding the liposomes can be widely adjusted in the range from room temperature to the phase transition temperature of each material, and extrusion can be repeated an appropriate number of times to make the liposome size uniform.

[0155] In addition, sonosensitive liposomes can be prepared using a microfluidic process. The organic solvents used in the microfluidic process can include, for example, one or more selected from the group consisting of dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether. According to one embodiment of the present invention, liposomes are prepared by a microfluidic process using ethanol and aqueous ammonium sulfate solution in a single-channel and a dual-channel microfluidic system, respectively.

[0156] In addition, the concentration of phospholipids dissolved in the organic solvent in the preparation method can be, for example, in the range of 1 to 300 mg / mL, 1 to 200 mg / mL, 1 to 100 mg / mL, 1 to 80 mg / mL, 1 to 70 mg / mL, 1 to 50 mg / mL, 1 to 30 mg / mL, 1 to 20 mg / mL, 1 to 10 mg / mL, 1 to 5 mg / mL, 1 to 3 mg / mL, 3 to 5 mg / mL, or 60 to 70 mg / mL, but the present invention is not limited thereto.

[0157] In addition, the flow rate of the microfluidic process in the preparation method can be, for example, in the range of 1 to 50 mL / min, 1 to 40 mL / min, 1 to 30 mL / min, 1 to 25 mL / min, 1 to 20 mL / min, or 1 to 15 mL / min, but the present invention is not limited thereto.

[0158] According to one embodiment of the present invention, liposomes are prepared with varying MSPC molar ratios and then the physical properties of the liposomes are compared. The results confirm that when liposomes are prepared using approximately 5 moles of MSPC, the liposomes have the highest stability while showing an excellent drug release rate (see Examples 2 and 7).

[0159] According to another embodiment of the present invention, liposomes are prepared with varying cholesterol molar ratios and then the physical properties of the liposomes are compared. The results confirm that cholesterol has a slight effect on sonosensitivity regardless of the ratio of cholesterol (see Example 3).

[0160] According to yet another embodiment of the present invention, liposomes optionally comprising DOPC or DOPE are prepared, and then the physical properties of the liposomes are compared. The results confirm that DOPE is more suitable for preparing sonosensitive liposomes (see Example 4).

[0161] According to another embodiment of the present invention, liposomes are prepared with varying molar ratios of DSPC, and then the physical properties of the liposomes are compared. The results confirm that the drug release rate by sonication decreases with an increase in the DSPC concentration (see Example 5).

[0162] According to another embodiment of the present invention, the rate of drug release from the liposomes of the present invention is determined according to the presence / absence of sonication and over time. The results confirm that the drug release rate of the sonication-irradiated group is significantly increased compared to the non-sonication-irradiated group, and the particle size distribution is maintained without any change (see Example 8).

[0163] According to another embodiment of the present invention, the cancer cell lethal effect of the liposomes of the present invention and commercially available liposome drugs is determined according to the presence / absence of sonotherapy. The results confirm that the sonosensitive liposomes of the present invention have a superior cancer cell lethal effect compared to commercially available liposome drugs (see Example 10).

[0164] According to yet another embodiment of the present invention, the breast cancer cell penetration effect of the liposomes of the present invention and commercially available liposome drugs is determined according to the presence / absence of sonotherapy. The results confirm that the sonosensitive liposomes of the present invention have a superior cancer cell penetration effect compared to commercially available liposome drugs (see Example 11).

[0165] Considering the functions in the present invention, the terms used in the present invention have been selected as general terms as widely as possible, but this may vary according to the intention of those skilled in the art or precedents, the emergence of new technologies, etc. In addition, in some cases, there are also terms arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the description of the present invention. Therefore, the terms used in the present invention should be defined based on the meaning of the terms and the entire content of the present invention, rather than simply the name of the terms.

[0166] Throughout the specification of the present invention, when any specific part is referred to as "comprising" any component, this means that it may further comprise other components rather than excluding other components, unless otherwise specified. In addition, when manufacturing and material tolerances inherent in the indicated meanings are intended to assist in understanding the present invention, the terms "about", "substantially", etc. used throughout the specification of the present invention are used for values that are at or close to the numerical values. Exact or absolute numbers are used to help prevent unfair use by unscrupulous infringers.

[0167] Throughout the specification of the present invention, the term "combinations thereof" included in a Markush-type expression refers to a mixture or combination of one or more selected from the group consisting of the components described in the Markush-type expression, and thus means that the mixture or combination includes one or more selected from the group consisting of the components. Detailed Description

[0169] Preferred embodiments of the present invention are described below to facilitate understanding of the present invention. However, it should be understood that the following embodiments are given only by way of illustration to more easily understand the present invention and are not intended to limit the present invention.

[0170] Examples

[0171] The materials listed in the following table were used in the examples.

[0172]

[0173] Example 1: Preparation of Liposomal Formulations

[0174] First, phospholipids including 32.1 mg of DSPC, 57.0 mg of DSPE-mPEG2000, 23.5 mg of cholesterol, 196.6 mg of DOPE, and 10 mg of lysophosphatidylcholine (1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine, MSPC) were dissolved in 5 mL of chloroform. The molar ratio of each material was 10:5:15:65:5. The phospholipid solution dissolved in chloroform was completely evaporated at 45 °C using a rotary evaporator to prepare a thin bilayer phospholipid film. Then, under stirring at 55 °C, the phospholipid film was hydrated in a 250 mM ammonium sulfate solution. The dispersed liposome solution was extruded at a temperature higher than room temperature using an extruder equipped with a polycarbonate membrane (mini extruder, Avanti). The extrusion was repeated until the size of the liposomes reached a size of approximately 150 nm.

[0175] After diluting the liposomes 10-fold, the particle size distribution of the liposomes was measured by a dynamic light scattering (DLS) analysis method using a Zetasizer Nano ZS (Malvern). The liposome solution with a regulated particle size distribution by extrusion was introduced through a dialysis membrane with a molecular weight cut-off (MWCO) of 15,000 daltons and dialyzed in a solution of 10% sucrose and 10 mM L-histidine for 12 hours.

[0176] To encapsulate doxorubicin in liposomes, doxorubicin was added to the dialyzed liposome solution and stirred at 37 °C and 200 to 300 rpm for 2 hours. The liposomes and doxorubicin were mixed at a weight ratio of 8:1. After stirring for 2 hours, unencapsulated doxorubicin was separated using size-exclusion chromatography (SEC) method. The absorbance of encapsulated doxorubicin and non-encapsulated doxorubicin was measured and quantified at a wavelength of 480 nm.

[0177] Example 2: Preparation of Liposomes According to the MSPC Ratio and Analysis of Drug Release Rate by Ultrasonication

[0178] Liposomes were prepared in the same manner as in Example 1. In this case, the molar ratio of MSPC was adjusted to 5 or 10, and the molar ratios of other components were fixed as described in Example 1. For the preparation of liposomes, the liposomes were hydrated by adjusting ammonium sulfate so that the final concentration of the liposomes reached 16 mg / mL, and the preparation process was carried out in the same manner as above. The particle size distribution and doxorubicin encapsulation efficiency of each liposome were also analyzed in the same manner as in Example 1.

[0179] To measure the level of doxorubicin released from liposomes by ultrasound, each liposome encapsulating doxorubicin was placed in the chamber of an ultrasonic device (UP200s, Hielscher) and ultrasound was applied at 29 kHz and 92 W / cm 2 for 1 minute. Analysis of the doxorubicin release rate sensitized by ultrasound was performed using liposomes, where 0.1 mg / mL doxorubicin was loaded based on doxorubicin. The released doxorubicin and the liposomes from which doxorubicin was released were separated using size-exclusion chromatography (SEC) method. Quantitative analysis of doxorubicin was carried out by measuring the absorbance at 480 nm. Table 1 and Figure 1 show the composition and particle size distribution of the corresponding liposomes, the encapsulation efficiency of doxorubicin, and the doxorubicin release rate by ultrasound.

[0180] [Table 1]

[0181] Particle size distribution of liposomes according to the MSPC molar ratio, doxorubicin encapsulation efficiency, and doxorubicin release rate by ultrasound

[0182]

[0183] As shown in Table 1 and Figure 1 as shown, the doxorubicin release rate sensitized by ultrasound increases with the decrease of hemolysis-PC. From these results, it can be seen that MSPC structurally increases the resistance of liposomes to ultrasound, thus inhibiting the release of drugs. Therefore, the inventors set the molar ratio of MSPC to 5 to perform the following examples.

[0184] Example 3: Analysis of Doxorubicin Release Rate by Ultrasound Sensitization According to Cholesterol Ratio

[0185] To analyze the effect of cholesterol on the release rate of doxorubicin by ultrasound-sensitized liposomes, liposomes were prepared with different cholesterol molar ratios. Liposomes were prepared by changing only the mass of cholesterol, but the molar ratios of DSPC, DSPE-mPEG2000, cholesterol, DOPE, and MSPC were based on 10:5:15:65:5 as described in Example 1. All the prepared liposomes were adjusted to the same concentration of 16 mg / mL and hydrated with ammonium sulfate (250 mM). The method for preparing liposomes, as well as the method for analyzing the particle size distribution of liposomes, doxorubicin encapsulation efficiency, and doxorubicin release rate by ultrasound sensitization, were carried out in the same manner as in Example 1.

[0186] Table 2 and Figure 2 shows the results of analyzing the effect of cholesterol on the doxorubicin release rate by ultrasound sensitization.

[0187] [Table 2]

[0188] The doxorubicin release rate through ultrasound-sensitized liposomes according to the change in cholesterol ratio

[0189]

[0190] As shown in Table 2 and Figure 2 as shown, it can be confirmed that when the ratio of cholesterol changes from 0 to 40, the ratio of cholesterol in the liposome composition has a slight effect on sonosensitivity, and regardless of the cholesterol ratio, the liposomes have a doxorubicin release rate of 60% or higher.

[0191] Example 4: Comparison of Doxorubicin Release Rates from Ultrasound-Sensitized DOPC and DOPE

[0192] Liposomes containing DOPC or DOPE with a molar ratio of 65% were prepared in the same manner as in Example 1, and the molar ratios of DSPC, DSPE-mPEG2000, cholesterol, and MSPC were fixed at 10, 5, 15, and 5. The liposomes were hydrated with ammonium sulfate (250 mM) to a concentration of 16 mg / mL. The methods for analyzing the particle size distribution, doxorubicin encapsulation efficiency, and doxorubicin release rate by ultrasound sensitization were carried out in the same manner as in Example 1.

[0193] Table 3 and Figure 3 shows the particle size distribution, drug encapsulation efficiency, and doxorubicin release rate by ultrasound sensitization of each liposome containing DOPC and DOPE.

[0194] [Table 3]

[0195] The particle size distribution, drug encapsulation efficiency, and doxorubicin release rate through ultrasound sensitization of each liposome containing DOPC and DOPE

[0196]

[0197] As shown in Table 3 and Figure 3 as shown, it was confirmed that no significant differences in particle size distribution and doxorubicin encapsulation efficiency were observed in each liposome containing DOPC and DOPE. However, for the ultrasound-mediated doxorubicin release rate, liposomes containing DOPE had a release rate of 60% or higher, while liposomes containing DOPC were less sensitive to ultrasound sensitization and thus had a release rate of approximately 20%. Therefore, it was confirmed that DOPE is more suitable as a component of the liposomes according to the present invention.

[0198] Example 5: Analysis of Doxorubicin Release from Ultrasound-Sensitized Liposomes According to DSPC Molar Ratio

[0199] Liposomes were prepared by fixing the molar ratios of DSPE-mPEG2000, cholesterol, DOPE, and MSPC at 5%, 15%, 65%, and 5%, respectively, and doubling and quadrupling the DSPC molar ratio based on the 10% DSPC prepared in Example 1. Then, the doxorubicin release rate through ultrasound sensitization was compared and analyzed.

[0200] The prepared liposomes were hydrated with ammonium sulfate (250 mM) such that the liposome concentration reached 16 mg / mL. The method of preparing and analyzing liposomes was carried out in the same manner as in Examples 1 and 2, and the drug release rate through ultrasound sensitization was analyzed.

[0201] Table 4 and Figure 4 show the particle size distribution, doxorubicin encapsulation efficiency, and doxorubicin release rate through ultrasound sensitization in each liposome based on the DSPC ratio.

[0202] [Table 4]

[0203] Particle size distribution of each liposome according to the DSPC molar ratio, doxorubicin encapsulation efficiency, and

[0204] Doxorubicin release rate in each liposome through ultrasound sensitization

[0205]

[0206] As shown in Table 4 and Figure 4 as shown, it was confirmed that regardless of the change in the DSPC ratio, there were no significant changes in the particle size distribution and doxorubicin encapsulation efficiency, and the liposomes had a particle size distribution of approximately 100 nm to 200 nm and a doxorubicin encapsulation efficiency of approximately 75% to 85%.

[0207] On the other hand, the release trend of doxorubicin sensitized by ultrasound was compared with the amount of DSPC added. The results confirmed that the release rate of doxorubicin by ultrasound decreased with the increase of the DSPC ratio.

[0208] Therefore, it was confirmed that a lower DSPC molar ratio was more suitable for preparing sonosensitive liposomes.

[0209] Example 6: Analysis of Doxorubicin Release Rate with Changes in Liposomal Composition Molar Ratio

[0210] Experiment

[0211] To analyze the release rate of doxorubicin sensitized by ultrasound for the composition constituting the liposome, liposomes were prepared using the composition ratios listed in Table 5. The method for preparing liposomes was carried out in the same manner as in Example 1, and the concentration of liposomes was adjusted to 16 mg / mL using ammonium sulfate (250 mM). Thereafter, the method for encapsulating doxorubicin, the encapsulation efficiency of doxorubicin, and the release rate of doxorubicin sensitized by ultrasound were also analyzed in the same manner as in Example 1.

[0212] [Table 5]

[0213] Analysis results of the release rate of doxorubicin sensitized by ultrasound according to the change in molar ratio in the liposome composition

[0214]

[0215] As shown in Table 5, it was confirmed that the release rate of doxorubicin sensitized by ultrasound tended to increase with the increase of the DOPE ratio, and cholesterol and MSPC improved the stability of liposomes while inhibiting the release of doxorubicin.

[0216] Example 7: Evaluation of Liposome Stability Based on MSPC Ratio

[0217] Evaluate the effect of MSPC on the stability of liposomes based on the MSPC content and MSPC molar ratio in liposomes. The composition of liposomes for stability evaluation is as described in Table 6. Liposomes were prepared in the same manner as in Example 1, and the target concentration of doxorubicin in the liposome solution was 0.40 mg / 1.0 mL solution.

[0218] [Table 6]

[0219] Liposome composition for stability evaluation

[0220]

[0221] To evaluate the stability of liposomes, each prepared liposome was mixed with fetal bovine serum (FBS) at a volume ratio of 1:3 and then stirred.

[0222] The conditions for evaluating the liposome stability are as follows: The mixed solution thus prepared is vortexed for 60 minutes at room temperature (20 to 25 °C). In this case, a portion of the solution is taken every 20 minutes, and then the amount of doxorubicin released from the liposome is measured using solid-phase extraction (Waters Oasis HLB 3CC). The pretreatment method is as Figure 5 shown. The elution solution is collected and analyzed according to the liquid chromatography analysis conditions shown in Table 7, and then the amount of released doxorubicin is analyzed.

[0223] [Table 7]

[0224] Liquid chromatography analysis conditions

[0225]

[0226]

[0227] As Figure 6 shown, it was confirmed that up to 30% or less of doxorubicin was released because the stability of the liposome was enhanced as the molar ratio of MSPC in the liposome composition increased, thereby suppressing the release of encapsulated doxorubicin. In addition, when MSPC was present at a concentration of 5 mol% or higher, the liposome was very stable.

[0228] Therefore, when the liposome contains MSPC, liposomes with excellent stability can be prepared, and the most stable liposomes can be prepared when the liposome contains 5 mol% MSPC.

[0229] Example 8: Experiment on Doxorubicin Release from Sonosensitizing Liposomes over Time

[0230] The release rate of doxorubicin over time was analyzed using the liposomes prepared in Example 1. In addition, to compare and evaluate the drug release trend by ultrasonic sensitization, samples were taken every 1 hour from the experimental group irradiated with ultrasound (US+) and the experimental group not irradiated with ultrasound (US-), and the released doxorubicin and the doxorubicin in the liposome were quantitatively analyzed. The release test was carried out under the conditions of 37 °C and 150 rpm. Ultrasonic-induced release was carried out by sampling at each time point, and the test solution was irradiated with ultrasound at a frequency of 29 kHz and an intensity of 92 W / cm 2 for 1 minute. Sampling and ultrasonic irradiation were repeated every hour. The released doxorubicin and liposomes were separated using size-exclusion chromatography (SEC) in the same manner as in Example 2. The encapsulated doxorubicin and non-encapsulated doxorubicin were quantified by measuring the absorbance at a wavelength of 480 nm.

[0231] Table 8 shows the composition, particle size distribution, and encapsulation efficiency of the liposomes used in this example. In addition, Table 9 and Figure 7Shows the release rate of doxorubicin from hypersensitive liposomes over time and the trend of comparison and evaluation of the doxorubicin release rate according to ultrasonic irradiation.

[0232] [Table 8]

[0233] Composition, particle size distribution and encapsulation efficiency of sonosensitive liposomes

[0234]

[0235] [Table 9]

[0236] Comparison and evaluation of the doxorubicin release rate from sonosensitive liposomes according to sonication or over time

[0237] (Ultrasound-induced release time: 1 minute of release per hour)

[0238]

[0239] As shown in Table 9 and Figure 7 As shown, the drug release trend of sonosensitive liposomes was compared with the experimental group without ultrasonic irradiation. When the sonosensitive liposomes were irradiated with ultrasound for 1 minute per hour, the sonosensitive liposomes had an improved release rate of approximately 20%. On the other hand, in the experimental group without ultrasonic irradiation, the ultrasound-sensitive liposomes had an initial doxorubicin release rate of approximately 15% and did not further show a doxorubicin release pattern. Through ultrasound-induced release, the sonosensitive liposomes had a drug release behavior of approximately 75% within 3 hours (a total of 3 times; 1 minute of irradiation per hour) and a doxorubicin release rate of approximately 80% within 6 hours (a total of 6 times; 1 minute of irradiation per hour). In addition, the particle size distribution before and after ultrasound-induced release was also analyzed. Therefore, it was confirmed that the particle size distribution remained unchanged, indicating that no particle fragmentation occurred due to the action of ultrasound.

[0240] Example 9: Preparation of Sonosensitizing Liposomes According to Hemolytic PC Type and Analysis of Sonosensitization Results

[0241] To analyze the changes in the physical properties and sonosensitivity of sonosensitive liposomes according to the type of hemolytic-PC, sonosensitive liposomes were prepared for each type of hemolytic-PC.

[0242] Five types of hemolytic-PC were selected and tested based on the type of hydrophobic chain. The types and component ratios of the selected hemolytic-PC are shown in Table 10 below.

[0243] [Table 10]

[0244] Types of hemolytic-PC and component ratios of sonosensitive liposomes

[0245]

[0246] To prepare sonosensitizing liposomes based on the acyl chain type of hemolytic-PC, weigh the lipid volume as specified in Table 10 and dissolve it in ethanol at 256 mg / mL at 60 °C. Then, add the lipid to a 250 mM ammonium sulfate solution and stir at 60 °C for 1 hour to prepare liposomes. In this case, the lipid concentration is 32 mg / mL. To reduce the liposome size, extrude the liposomes using a high-temperature extruder. By passing the liposomes through a polycarbonate filter of the extruder, the liposome size is gradually reduced from a 0.8-μm pore size to a 0.4-μm or 0.2-μm pore size, and this process is repeated until the liposome size reaches 100 nm to 200 nm. Use a PD-10 column to replace the mixed solution of 10% sucrose and 10 mM histidine used as the solvent for the liposome solution with ammonium sulfate.

[0247] Next, to encapsulate doxorubicin, doxorubicin and liposomes are mixed at a mass ratio of 1:8 and then stirred at 37 °C for 1 hour. Use a PD-10 column to separate the unencapsulated doxorubicin. Analyze the particle size distribution of each prepared sonosensitizing liposome and the quantification of doxorubicin in the same manner as in Examples 1 and 2.

[0248] [Table 11]

[0249] Doxorubicin release rate of sonosensitizing liposomes according to the hemolytic-PC type

[0250]

[0251] As shown in Table 11, according to the type of hemolytic-PC, the doxorubicin release rate has a similar trend of 55% to 65%. Therefore, it is judged that the change in the tail part of hemolytic-PC will not affect the doxorubicin release rate through sonosensitivity.

[0252] Example 10: Comparison and Analysis of the Cancer Cell Lethal Effect of Sonosensitizing Liposomes

[0253] To compare and analyze the cell death effect of the sonosensitive liposomes according to the presence / absence of ultrasonic irradiation, the sonosensitive liposomes of Example 1 were used to compare and analyze the cancer cell lethal effect according to the presence / absence of ultrasonic irradiation. The MDA-MB-231 cell line, which is a triple-negative breast cancer cell line, was used as the cancer cells. The MDA-MB-231 cell line was inoculated into each well of a 96-well plate at a concentration of 10,000 cells / mL and then cultured overnight. The cell culture was cultured in a culture broth containing 10% fetal bovine serum (FBS) and 1% antibiotic in Dulbecco's Modified Eagle Medium (DMEM). The culture broth was removed from the wells in which the cells were cultured overnight, replaced with DMEM without FBS, and then cultured for 1 hour. Then, the sonosensitive liposomes were added thereto and cultured for 3 hours. To compare and analyze cancer cell death according to ultrasonic irradiation, each designated well was treated with sonosensitive liposomes irradiated with ultrasound (ultrasonic liposomes (US+)) and ultrasound-sensitive liposomes not irradiated with ultrasound (ultrasonic liposomes (US-)). Each well was treated with doxorubicin at a concentration of 2, 4, 6, 8, or 10 μg / mL. The commercially available doxorubicin liposome drug (liposomal doxorubicin HCl) was used as a control, and each well was treated with Caelyx in the same manner as the sonosensitive liposomes. After treating the wells with each liposome, the wells were cultured for 3 hours, the treated samples present in each well were removed, and then the wells were washed three times with PBS. Then, DMEM containing 10% FBS and 1% antibiotic was added thereto again, and the cells were cultured. After the cells were cultured for 72 hours, the culture medium present in each well was removed, 20 μL of MTT reagent at a concentration of 2 mg / mL was added, and the cells were cultured for 3 hours. The formazan formed by adding 200 μL of DMSO to each well was used, and the absorbance at a wavelength of 540 nm was measured using an ELISA reader to compare and analyze the cancer cell lethal effect.

[0254] As a result, as Figure 9 shown, in the case of the experimental group without ultrasonic irradiation, both the sonosensitive liposomes and both had a poor cell death effect on cancer cells. Only doxorubicin had a cancer cell lethal effect because it penetrated into the cells.

[0255] On the other hand, in the experimental group with ultrasonic irradiation, had a cancer cell lethal effect similar to that of the experimental group without ultrasonic irradiation, while the sonosensitive liposomes of the present invention had an increased cancer cell lethal effect with the release of the encapsulated doxorubicin, indicating that drug release could be effectively induced by ultrasound. Therefore, it was confirmed that the liposomes of the present invention showed excellent sensitivity to ultrasound compared to existing commercially available liposome drugs.

[0256] Example 11: Analysis of the Cell Penetration Effect of Sonosensitizing Liposomes

[0257] To Analysis evaluate the cell penetration effect of the sonosensitive liposomes, the cell penetration effect of the sonosensitive liposomes in Example 1 was compared and analyzed with that of . MDA-MB-231 cell line was seeded in an 8-well cell culture chamber at a concentration of 30,000 cells / mL and cultured overnight. The cell culture broth of Example 9 was used. After the cells were cultured overnight, the cell culture broth present in each well was removed, and the cells were cultured in a cell culture broth without FBS for 1 hour. Thereafter, sonosensitive liposomes and

[0258] As a result, as Figure 10 shown, neither nor liposomes without sonication penetrated into the cells. However, in the case of the experimental group with sonication, doxorubicin was released from the sonosensitive liposomes and penetrated into the cells, indicating its excellent sonosensitivity. In addition, it was confirmed that doxorubicin penetrated into the cells within 3 hours.

[0259] Example 12: Preparation and Evaluation of Sonosensitizing Liposomes Using a Microfluidic Process and Ultrasonic Sensitization Experiment

[0260] To prepare sonosensitive liposomes using a microfluidic process, a phospholipid component (including DSPC, DSPE-mPEG2000, cholesterol, DOPE, and MSPC with molar ratios of 10, 5, 15, 65, and 5) was dissolved in ethanol. To dissolve completely, the phospholipid component was stored in an oven at 75 °C for 10 minutes such that the phospholipid component was dissolved in a clear solution. The solution was prepared at concentrations of 2, 4, 8, 16, and 64 mg / mL depending on the experimental group.

[0261] The microfluidic process consists of two channels, one channel being the path for applying the phospholipids dissolved in ethanol and the other channel being the path for applying 250 mM ammonium sulfate.

[0262] For the administration ratio in each channel, the experiments described in Table 12 were conducted. In the case of the experimental group, the total flow rate was adjusted to 3, 12, and 21 mL / min. The liposome solution discharged through the channel was dialyzed through a permeable membrane with a molecular weight cut-off (MWCO) of 15,000 Da for 24 hours. In this case, the solution was dialyzed in a solution containing 10% sucrose and 10 mM L-histidine. After the dialysis process, the method for analyzing the particle size was carried out in the same manner as in Example 1. Table 12 lists the molar ratios of the phospholipid components of the liposomes and the experimental conditions.

[0263] [Table 12]

[0264] Preparation composition, experimental conditions, and particle size distribution of liposomes using a microfluidic process

[0265]

[0266] As shown in Table 12, it was confirmed that most liposomes had a particle size distribution of 100 to 200 nm. It was confirmed that the factors regulating the liposome size included the phospholipid concentration dissolved in ethanol, the total flow rate, and the volume ratio of ethanol and ammonium sulfate. As the difference in the volume ratio increased, the particle size distribution tended to become smaller. In addition, as the total flow rate increased and the phospholipid concentration decreased, the particle size distribution tended to decrease.

[0267] Example 13: Preparation of Liposomes by Ethanol Addition Method

[0268] Sonosensitive liposomes were prepared by the ethanol addition method using the lipid composition listed in Table 13 to prepare sonosensitive liposomes. Each lipid mixture weighed according to the molar ratios listed in Table 13 was dissolved in ethanol at a concentration of 256 mg / mL at 70 °C. The lipid solution dissolved in a completely clear solution was mixed with a 250 mM aqueous ammonium sulfate solution. In this case, the concentration of the lipid was 32 mg / mL, and the lipid solution was stirred for 1 hour while maintaining the mixing temperature at 60 °C. The mixed lipid solution was homogenized by a high-temperature extruder (LIPEX, Evonik). When using a high-temperature extruder, the size of the liposomes was gradually reduced from a 0.8-μm pore size to a 0.4-μm or 0.2-μm pore size through a polycarbonate filter in the extruder to prepare liposomes. This process was repeated until the size of the prepared liposomes reached 100 nm to 200 nm.

[0269] The prepared liposome solution was replaced with a mixed solution of 10% sucrose and 10 mM histidine, and size-exclusion chromatography (SEC) or size-exclusion chromatography method was carried out using FPLC. SEC was carried out using a high-preparation 26 / 10 column at a flow rate of 10 mL / min.

[0270] To encapsulate doxorubicin, doxorubicin and liposomes having the compositions listed in Table 11 were mixed at a mass ratio of 1:8 and stirred at 37 °C for 1 hour. Subsequently, unencapsulated doxorubicin was separated using a size-exclusion chromatography method.

[0271] Example 14: Sonosensitization Test of Liposomes Prepared by Ethanol Addition Method

[0272] A sonosensitization test was performed on doxorubicin-encapsulating liposomes prepared by the ethanol addition method using high-intensity focused ultrasound. The compositions of the prepared liposomes are shown in Table 13. The ultrasound equipment for the sonosensitization test was used in the same manner as described in Example 2. The conditions for releasing ultrasound during the doxorubicin sonosensitization test were as follows: using a frequency of 29 kHz and 92 W / cm 2 The ultrasound intensity released doxorubicin for 60 seconds. The released doxorubicin was purified by the SEC method and quantified using the absorbance at a wavelength of 475 nm.

[0273] [Table 13]

[0274] Physical properties and sonosensitization results of liposomes prepared by the ethanol addition method

[0275]

[0276] Example 15: Pharmacokinetic (pK) test according to the MSPC ratio

[0277] Among the sonosensitizing liposomes used in Example 7, liposomes containing 1%, 5%, and 7% molar ratios of MSPC were selected, and a pK test was performed using Sprague-Dawley rats. The compositions of the sonosensitizing liposomes for the pK test are listed in Table 14.

[0278] [Table 14]

[0279] Liposome compositions for pK evaluation according to the MSPC ratio

[0280]

[0281] Each liposome was intravenously injected into Sprague-Dawley rats at a dose of 2 mg / kg, and blood was collected from the jugular vein of each rat. Blood collection was performed at time points of 30 minutes, 1 hour, 4 hours, 12 hours, 24 hours, and 48 hours, and an anticoagulant (i.e., sodium heparin) was used to prevent the collected blood from coagulating. Then, the collected blood was centrifuged at 12,000 rpm for 2 minutes to separate plasma. Quantitative analysis of doxorubicin was performed using LC-MS / MS, and the change in pK was evaluated according to the ratio of MSPC.

[0282] Table 15 and Figure 11The pK results according to the MSPC ratio are shown. The results confirmed that the volume of the sonosensitive liposomes present in vivo increased with the increase in the MSPC ratio. When the MSPC content was 1%, doxorubicin with a concentration of 5.1 ± 1.5 ng / mL was detected after 48 hours. When the MSPC contents were 5% and 7%, doxorubicin with concentrations of 4.4 ± 10.3 and 35.6 ± 17.9 were detected, respectively.

[0283] In addition, when the MSPC contents were 1%, 5%, and 7%, the in vivo half-lives were found to be 4.0 ± 0.1 hours, 6.4 ± 0.9 hours, and 5.7 ± 0.7 hours, respectively. The results indicated that since the in vivo stability was improved with the increase in the MSPC ratio, the in vivo stability was maintained for a long time in the blood, and MSPC enhanced the blood stability of the sonosensitive liposomes.

[0284] [Table 15]

[0285] pK test results according to the MSPC ratio (MSPC - 1%, 5%, and 7%)

[0286]

[0287] The above description of the present invention is given only by way of illustration. Therefore, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or basic characteristics of the present invention. Therefore, it should be understood that the above embodiments are for illustrative purposes only and are not restrictive in all aspects.

[0288] Industrial Applicability

[0289] It was confirmed that the sonosensitive liposomes according to the present invention have a high encapsulation efficiency, excellent sonosensitivity and liposome stability, and compared with existing commercial drugs, show excellent effects of improving the drug release rate by sonication, as well as cancer cell lethal effect and cancer cell penetration effect. Therefore, the sonosensitive liposomes according to the present invention improve the delivery efficiency of the encapsulated drug and have an improved drug delivery effect when used in combination with sonication to target cancer cells. Therefore, the sonosensitive liposomes according to the present invention are expected to show various functions in the fields of cancer treatment and drug delivery carriers. Therefore, the sonosensitive liposomes of the present invention are industrially applicable.

Claims

1. A sound-sensitive liposomal preparation, which comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated with methoxypoly(ethylene glycol 2000) (DSPE-mPEG2000), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and lysophosphatidylcholine (lyso-PC), wherein the DSPC, the DSPE-mPEG2000, the DOPE, the cholesterol and the lyso-PC are included in a molar ratio of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20 (mol %).

2. The sound-sensitive liposomal preparation according to claim 1, wherein the DSPC is included in a dry weight of 0.1 to 50% based on the total weight of the liposomal preparation. The DSPE-mPEG2000 is included in a dry weight of 3 to 50% based on the total weight of the liposomal preparation. The DOPE is included in a dry weight of 1 to 80% based on the total weight of the liposomal preparation. The cholesterol is included in a dry weight of 0.05 to 40% based on the total weight of the liposomal preparation, and The lyso-PC is included in a dry weight of 0.5 to 10% based on the total weight of the liposomal preparation.

3. The sound-sensitive liposomal preparation according to claim 1, wherein the liposomal membrane of the sound-sensitive liposomal preparation is composed of DSPC, DSPE-mPEG2000, DOPE, cholesterol and lysophospholipase-PC.

4. The sound-sensitive liposomal preparation according to claim 1, wherein a drug is encapsulated in the sound-sensitive liposomal preparation.

5. The sound-sensitive liposomal preparation according to claim 4, wherein the drug is an anticancer drug.

6. The sound-sensitive liposomal preparation according to claim 1, wherein the sound-sensitive liposomal preparation has one or more of the following characteristics: i. The particle size is 100 nm to 200 nm; and ii. The drug encapsulation efficiency is 50% to 100%.

7. A pharmaceutical composition for preventing or treating cancer, which comprises the sound-sensitive liposomal preparation according to claim 1.

8. The pharmaceutical composition according to claim 7, wherein the cancer comprises one or more selected from the group consisting of breast cancer, colon cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, skin cancer, head cancer, neck cancer, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, lymphocytic lymphoma, bladder cancer, kidney cancer or ureteral cancer, and CNS tumors.

9. The pharmaceutical composition according to claim 7, wherein the cancer comprises one or more selected from the group consisting of small cell lung cancer, pancreatic cancer, cutaneous melanoma, colorectal cancer, endometrial cancer, cervical cancer, Hodgkin's disease, thyroid cancer, parathyroid cancer, adrenal cancer, chronic or acute leukemia, renal cell carcinoma, renal pelvic carcinoma, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma.

10. A composition for drug delivery, which comprises liposomes encapsulating a drug as an active ingredient, wherein the liposomes comprise DSPC, DSPE-mPEG2000, DOPE, cholesterol and lysophosphatidylcholine, and wherein the DSPC, the DSPE-mPEG2000, the DOPE, the cholesterol and the lysophosphatidylcholine are included in a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20.

11. The composition for drug delivery according to claim 10, wherein the drug is an anti-cancer drug.

12. The composition for drug delivery according to claim 11, wherein the anti-cancer drug comprises one or more selected from the group consisting of doxorubicin, paclitaxel, docetaxel, cisplatin, Gleevec, 5-fluorouracil (5-FU), tamoxifen, carboplatin, topotecan, belotecan, imatinib, irinotecan, floxuridine, vinorelbine, gemcitabine, leuprorelin, flutamide, zoledronate, methotrexate, camptothecin, vincristine, hydroxyurea, streptozotocin, oxaliplatin, valrubicin, mechlorethamine, chlorambucil, busulfan, doxifluridine, vinblastine, mitomycin, everolimus and mitoxantrone.

13. The composition for drug delivery according to claim 10, wherein the drug is one or more selected from folinic acid, retinoic acid and prednisone.

14. A method for preparing the sonosensitive liposomes as claimed in claim 1, the method comprising the following preparation process: (a) Dissolving DSPC, DSPE-mPEG2000, DOPE, cholesterol and lysophosphatidylcholine in a first organic solvent; (b) Evaporating the organic solvent to prepare a liposome membrane; (c) Hydrating the liposome membrane in an aqueous solution and stirring the liposome membrane solution; and (d) Extruding the liposome membrane solution through an extruder, wherein the DSPC, the DSPE, the DOPE, the cholesterol and the lysophosphatidylcholine are dissolved in a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20.

15. A method for preparing the sonosensitive liposomes as claimed in claim 1, comprising the following preparation process: (a) Dissolving DSPC, DSPE-mPEG2000, DOPE, cholesterol and lysophosphatidylcholine in ethanol; (b) Hydrating the ethanol solution prepared in step (a) in an aqueous solution and stirring the ethanol solution; and (c) Extruding the ethanol solution through an extruder, wherein the DSPC, the DSPE, the DOPE, the cholesterol and the lysophosphatidylcholine are dissolved in a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20.

16. A method for preparing the sonosensitive liposomes as claimed in claim 1, comprising the following preparation process: (a) Dissolving DSPC, DSPE-mPEG2000, DOPE, cholesterol and lysophosphatidylcholine in a first organic solvent; (b) Mix the first organic solvent solution prepared in step (a) with an aqueous solution while changing its flow rate using the channels of the microfluidic system; and (c) Remove the organic solvent, wherein the DSPC, the DSPE, the DOPE, the cholesterol, and the lyso-PC are dissolved at a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20.

17. The method according to claim 14 or 16, wherein the first organic solvent comprises one or more selected from the group consisting of dimethylacetamide, dimethylformamide, dimethyl sulfoxide, chloroform, methanol, ethanol, and ether.

18. Use of the sonosensitive liposomal preparation according to claim 1 in the preparation of a cancer therapeutic agent.

19. Use of the liposome encapsulating a drug in the preparation of a drug delivery carrier, wherein the liposome comprises DSPC, DSPE-mPEG2000, DOPE, cholesterol, and lyso-PC, wherein the DSPC, the DSPE, the DOPE, the cholesterol, and the lyso-PC are included at a molar ratio (mol%) of 1 to 50:1 to 10:5 to 80:0.1 to 50:0.1 to 20.

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