Method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation of low intensity pulsed ultrasound (LIPUS) under tumor microenvironment, and combination regimen of gemcitabine and cisplatin with low intensity pulsed ultrasound for inhibiting cholangiocarcinoma growth

Abstract

The present invention relates to a method using low intensity pulsed ultrasound (LIPUS) in order to improve drug delivery efficiency in a hypoxic tumor microenvironment (TME) of cholangiocarcinoma (CCA), and more specifically, to a method for confirming the drug permeability and cancer cell apoptosis of cholangiocarcinoma through the irradiation of low intensity pulsed ultrasound (LIPUS) under a tumor microenvironment, and a combination regimen, of gemcitabine and cisplatin with low-intensity pulsed ultrasound, for inhibiting cholangiocarcinoma growth. To this end, provided is the method for confirming the drug permeability and cancer cell apoptosis of cholangiocarcinoma through the irradiation of low intensity pulsed ultrasound (LIPUS) under a tumor microenvironment, the method comprising: a step (S100) for administering a drug and a fluorescent material to cholangiocarcinoma (CCA) under a tumor microenvironment; a step (S120) for allowing a predetermined time to elapse; a step (S140) for irradiating the cholangiocarcinoma (CCA) low intensity pulsed ultrasound (LIPUS); a step (S160) for acquiring a 3D image by performing tissue-clearing on the cholangiocarcinoma (CCA); and a step (S180) for confirming the in vivo drug permeability of a drug and a fluorescent material from the 3D image.

Description

Method for confirming drug permeability and cancer cell apoptosis in cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in the tumor microenvironment, and combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibition of cholangiocarcinoma growth

[0001] The present invention relates to a method for using low-intensity pulsed ultrasound (LIPUS) to improve drug permeation efficiency in the hypoxic tumor microenvironment (TME) of cholangiocarcinoma (CCA). More specifically, the invention relates to a method for confirming the intratumoral anticancer drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) under the tumor microenvironment, and to measuring the improvement of the anticancer effect accordingly.

[0002] Generally, the tumor microenvironment (TME) in which tumor cells exist plays a crucial role in tumor formation and progression within tumor tissue. The TME consists of tumor cells, tumor stromal cells (including cancer-associated fibroblasts (CAF), tumor-associated macrophages (TAM), and T or B lymphocytes), the extracellular matrix (ECM), and secreted proteins. Tumor cells within the TME dynamically interact with stromal cells and the ECM, contributing to the promotion of tumor evolution and metastasis. The TME is characterized by irregular vascular networks, reduced blood flow, unequal perfusion, elevated interstitial fluid pressure (IFP), impaired lymphatic drainage, heterogeneous tumor vascular permeability, abundant stromal cells, and dense ECM and tumor cells. These pathological TME conditions impede clinical drug delivery to the tumor, such as systemically administered chemotherapy drugs and their carriers. Insufficient drug concentrations within the tumor interfere with the efficacy of anticancer agents. Overcoming these problems is important for cancer treatment.

[0003] Recently, many innovative applications of ultrasound therapy have been reported in the medical field. Ultrasound operates by transmitting sound waves through the body. In particular, low-intensity pulsed ultrasound (LIPUS) is one of the standard ultrasound themes attracting attention due to its improved safety. Applying low-intensity ultrasound in a pulsed manner results in relatively fewer thermal effects and side effects on biological tissues, which is less burdensome than high-intensity and continuous energy delivery. It has been reported that low-intensity pulsed ultrasound (LIPUS) can stimulate the delivery of anticancer drugs to target cells or tissues.

[0004] Furthermore, tumor hypoxia has recently emerged as a major problem in diagnosis and treatment, as reduced oxygen tension hinders the delivery of chemotherapy drugs to areas characterized by abnormal vascular structures and malignant gene expression.

[0005] Prior art literature

[0006] Patent documents

[0007] (Patent Document 1) 1. Republic of Korea Patent Registration No. 10-1940950 (Method for diagnosing cholangiocarcinoma through bacterial metagenomic analysis),

[0008] (Patent Document 2) 2. Republic of Korea Patent Registration No. 10-1395823 (Method for anticancer treatment using ultrasound and device for anticancer treatment).

[0009] Accordingly, the present invention has been devised to solve the above-mentioned problems, and the problem to be solved by the present invention is to provide a method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment where drug permeation efficiency into the hypoxic region tumor microenvironment (TME) can be evaluated using clear tumor imaging in cholangiocarcinoma (CCA), and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

[0010] However, the technical problems to be solved by the present invention are not limited to those mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0011] To achieve the above technical objectives, a method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting cholangiocarcinoma growth are provided, characterized by comprising the steps of: administering a drug and a fluorescent substance to cholangiocarcinoma (CCA) in a tumor microenvironment (S100); allowing a predetermined time to elapse (S120); irradiating the cholangiocarcinoma (CCA) with low-intensity pulsed ultrasound (LIPUS) (S140); performing tissue-enhanced clarity on the cholangiocarcinoma (CCA) to obtain a 3D image (S160); and confirming the drug permeability of the drug and fluorescent substance in vivo from the 3D image (S180).

[0012] In addition, cholangiocarcinoma (CCA) may be cholangiocarcinoma (CCA) with hypoxic regions.

[0013] In addition, the low-intensity pulsed ultrasound (LIPUS) has an ISPTA intensity of 0.1 to 0.7 W / cm² and a duty cycle of 5 to 45%, preferably with an ISPTA intensity of 0.5 W / cm² and a duty cycle of 45%. Optionally, the ISPTA intensity may be 0.7 W / cm² and the duty cycle may be 22%.

[0014] In addition, the prescribed time is 20 minutes to 24 hours.

[0015] In addition, anticancer drugs may include gemcitabine and / or cisplatin.

[0016] In addition, the fluorescent material is fluorescent ruthenium and liposome nanoparticles encapsulating ruthenium, and specifically, it may be a fluorescent ruthenium-liposome complex.

[0017] In addition, in the drug permeability verification step (S180), the drug permeability can be calculated and verified based on pixel information regarding the penetration depth and distribution of the drug and fluorescent material.

[0018] Additionally, the method may further include a step (S200) of confirming the distribution of apoptosis within cholangiocarcinoma (CCA) from a 3D image.

[0019] In addition, the growth of cholangiocarcinoma (CCA) may be inhibited by the administration of gemcitabine and cisplatin and irradiation with low-intensity pulsed ultrasound (LIPUS).

[0020] According to one embodiment of the present invention, the cell membrane permeability of anticancer agents and fluorescent substances can be increased. That is, the periodic pressure generated by low-intensity pulsed ultrasound induces vibrations to temporarily open the cell membrane, thereby promoting the absorption of anticancer agents.

[0021] Furthermore, low-intensity pulsed ultrasound is effective in improving fluid flow within tissues. This is because the compression and rarefaction patterns formed by pressure waves, combined with unidirectional flow, cause movement in a constant, single direction. This unidirectional flow can increase the distance and speed at which drug particles travel to target areas, including hypoxic regions within the tumor.

[0022] In addition, according to the present invention, high-resolution 3D images can be obtained through tissue clarity, and there is an advantage in being able to quantitatively calculate and confirm the penetration depth, distribution, and death of cancer cells that have absorbed the anticancer drug and fluorescent substance caused by low-intensity pulsed ultrasound (LIPUS) irradiation.

[0023] In addition, when cholangiocarcinoma (CCA) is treated with the anticancer agents gemstabine and cisplatin combined with low-intensity pulsed ultrasound (LIPUS) according to the present invention, there is an effect of inhibiting tumor growth.

[0024] In addition, the present invention can provide a safe and economical cancer treatment technology compared to traditional chemotherapy or drug delivery systems (DDS).

[0025] However, the effects obtainable from the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0026] The following drawings attached to this specification illustrate preferred embodiments of the present invention and serve to further enhance understanding of the technical concept of the present invention together with the detailed description of the invention provided below; therefore, the present invention should not be interpreted as being limited only to the matters described in such drawings.

[0027] Figure 1 is a schematic diagram showing the process of treating mice with cholangiocarcinoma (CCA) with low-intensity pulsed ultrasound (LIPUS) under various duty cycles and intensities (ISPTA) after administering a drug.

[0028] FIG. 2 is an example of 3D clear tumor imaging showing the abnormal vascular network and spatial distribution of low-intensity pulsed ultrasound (LIPUS)-mediated drug-induced apoptosis cancer cell death in cholangiocarcinoma (CCA) according to the present invention.

[0029] FIG. 3 is a graph showing the number of apoptotic cancer cells induced by treatment with gemcitabine and cisplatin (Gem / Cis) combined with low-intensity pulsed ultrasound (LIPUS) in relation to distance from blood vessels under various duty cycles (5%, 22%, 45%) and treatment delays (20 minutes and 24 hours) according to the present invention.

[0030] FIG. 4 shows ISPTA 0.1 W / cm² at 24 hours after drug injection according to the present invention. 2 , 0.3W / cm 2 , 0.5W / cm 2 , 0.7W / cm 2 An example of a 3D clear tumor image obtained from a cholangiocarcinoma (CCA) mouse that received low-intensity pulsed ultrasound (LIPUS) under the conditions of

[0031] Figure 5 shows various ISPTA (0.1W / cm²) 2 , 0.3W / cm 2 , 0.5W / cm 2 , 0.7W / cm 2 A graph showing the number of apoptotic cancer cells induced by treatment with gemcitabine and cisplatin (Gem / Cis) combined with low-intensity pulsed ultrasound (LIPUS) in relation to distance from blood vessels under ) conditions,

[0032] FIG. 6 is an example of a 3D clear tumor image obtained from a cholangiocarcinoma (CCA) mouse that received low-intensity pulsed ultrasound (LIPUS) at duty cycles of 5%, 22%, and 45% 24 hours after injection of liposome nanoparticles (Lip@Ru) according to the present invention.

[0033] Figure 7 is a graph showing the number of cancer cells that absorbed ruthenium (Ru) by the application of low-intensity pulsed ultrasound (LIPUS) in relation to the distance from blood vessels under various duty cycles (5%, 22%, 45%).

[0034] FIG. 8 shows ISPTA 0.1 W / cm² at 24 hours after injection of liposome nanoparticles (Lip@Ru) according to the present invention. 2 , 0.3W / cm 2 , 0.5W / cm 2 , 0.7W / cm 2 An example of a 3D clear tumor image obtained from a cholangiocarcinoma (CCA) mouse that received low-intensity pulsed ultrasound (LIPUS) under the conditions of

[0035] Fig. 9 shows various ISPTA (0.1W / cm²) 2 , 0.3W / cm 2, 0.5W / cm 2 , 0.7W / cm 2 A graph showing the number of cancer cells that absorbed ruthenium (Ru) by the application of low-intensity pulsed ultrasound (LIPUS) in relation to the distance from blood vessels under ) conditions,

[0036] FIG. 10 is a graph showing the ratio of cancer cells expressing Hif-1α (Hypoxia-Inducible Factor 1-alpha) and apoptotic Hif-1α cancer cells in relation to the distance from a blood vessel in an embodiment according to the present invention.

[0037] FIG. 11 is an example of a 3D clear tumor image showing the spatial distribution of apoptotic cancer cells in a hypoxic region and under hypoxic conditions in cholangiocarcinoma (CCA) after tissue clearing according to the present invention.

[0038] FIGS. 12 to 15 are graphs showing the results of a statistical analysis of low-intensity pulsed ultrasound (LIPUS)-mediated Gem / Cis-induced apoptosis and Lip@Ru uptake in various duty cycles and ISPTA according to the present invention.

[0039] FIGS. 16 and 17 are schematic diagrams and graphs showing tumor growth profiles by combination therapy of an anticancer agent and low-intensity pulsed ultrasound (LIPUS) in cholangiocarcinoma (CCA) according to the present invention.

[0040] FIG. 18 is a flowchart schematically illustrating a method for confirming drug permeability of cholangiocarcinoma and a method for confirming apoptosis of cancer cells by irradiation with low-intensity pulsed ultrasound (LIPUS) under a tumor microenvironment according to an embodiment of the present invention.

[0041] Below, with reference to the attached drawings, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, since the description of the present invention is merely an example for structural or functional explanation, the scope of the present invention should not be interpreted as being limited by the embodiments described in the text. That is, since the embodiments are subject to various modifications and may take various forms, the scope of the present invention should be understood to include equivalents capable of realizing the technical concept. Furthermore, the objectives or effects presented in the present invention do not imply that a specific embodiment must include all of them or only such effects; therefore, the scope of the present invention should not be understood as being limited by them.

[0042] The meaning of the terms described in this invention should be understood as follows.

[0043] Terms such as "first" and "second" are intended to distinguish one component from another, and the scope of rights shall not be limited by these terms. For example, the first component may be named the second component, and similarly, the second component may be named the first component. When a component is referred to as being "connected" to another component, it should be understood that it may be directly connected to that other component, or that there may be other components in between. Conversely, when a component is referred to as being "directly connected" to another component, it should be understood that there are no other components in between. Meanwhile, other expressions describing the relationship between components, such as "between" and "exactly between," or "adjacent to" and "directly adjacent to," shall be interpreted in the same manner.

[0044] A singular expression should be understood to include a plural expression unless the context clearly indicates otherwise, and terms such as "include" or "have" are intended to specify the existence of the set-up features, numbers, steps, actions, components, parts, or combinations thereof, and should be understood not to preclude the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0045] Unless otherwise defined, all terms used herein have the same meaning as generally understood by those skilled in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having meanings consistent with the context of the relevant technology and should not be interpreted as having an ideal or overly formal meaning unless explicitly defined in this invention.

[0046] Method to check drug permeability

[0047] Figure 1 is a schematic diagram showing the process of performing low-intensity pulsed ultrasound (LIPUS) under various duty cycles and intensities (ISPTA) on mice with cholangiocarcinoma (CCA) after administering drugs. As shown in Figure 1, cholangiocarcinoma (CCA) originates in the bile ducts and is the second most common primary liver cancer. CCA has a particularly poor prognosis, as it is often diagnosed at an advanced stage; 75% of patients die within one year of diagnosis, and the 5-year survival rate is less than 5%. Although surgical resection is the only long-term treatment method, most cases of CCA cannot be completely removed surgically. Although combination therapy with gemcitabine and cisplatin is used as a first-line treatment for advanced or unresectable CCA, this standard chemotherapy regimen provides only minimal improvement in progression-free survival.

[0048] FIG. 18 is a flowchart schematically illustrating a method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) under a tumor microenvironment according to an embodiment of the present invention. As shown in FIG. 18, a specific drug and a fluorescent substance are administered to hypoxic cholangiocarcinoma (CCA) under a tumor microenvironment (S100). Here, the specific drug is an anticancer agent comprising gemcitabine and / or cisplatin, and the fluorescent substance is a liposomal nanoparticle, specifically a fluorescent ruthenium (Ruthenium II, Ru) complex. These drugs and fluorescent substances are schematically shown in 3D clear tumor imaging in relation to tumor cell density, irregular blood vessels, and hypoxic regions within the tumor microenvironment (TME).

[0049] Next, allow a predetermined time to elapse (S120). At this time, the predetermined time may be 20 minutes or 24 hours after drug administration. 20 minutes takes into account the half-life of gemcitabine and cisplatin (Gem / Cis).

[0050] Next, low-intensity pulsed ultrasound (LIPUS) is applied to the cholangiocarcinoma (CCA) (S140). The low-intensity pulsed ultrasound (LIPUS) has an ISPTA intensity of 0.1 to 0.7 W / cm² 2 and, the duty cycle is 5 to 45%, and preferably, the ISPTA strength is 0.5 W / cm 2 And, the duty cycle is 45%. And, optionally, the ISPTA intensity is 0.7 W / cm² 2 And, the duty cycle can be 22%.

[0051] Next, tissue clarification (CLARITY) is performed on cholangiocarcinoma (CCA) to obtain high-resolution 3D images (S160). Tissue clarification (CLARITY) is a technique that enables spatial imaging analysis of biological structures without the need to cut or reconstruct the tissue. The presence of a dense lipid bilayer within animal tissue creates diffusion barriers and light scattering, hindering light and macromolecules from penetrating deeper tissue layers. However, tissue clarification (CLARITY) enables tissue transparency by stabilizing the tissue with a water-soluble hydrogel following a non-destructive lipid removal process, allowing high-resolution 3D images to be obtained from uncut tissue. Through these 3D images, the distribution of drugs and hypoxic regions can be identified based on their relationship with blood vessels within the tumor.

[0052] Next, the drug permeability (e.g., penetration depth and distribution) of the anticancer agent and liposome nanoparticles in vivo is determined from the acquired 3D image (S180). The drug permeability can be calculated and determined by a dedicated computer program based on pixel information regarding the penetration depth and distribution of the drug and fluorescent material.

[0053] Experimental results

[0054] FIG. 2 is an example of 3D clear tumor imaging showing the abnormal vascular network and spatial distribution of low-intensity pulsed ultrasound (LIPUS)-mediated drug-induced apoptosis cancer cell death in cholangiocarcinoma (CCA) according to the present invention. As shown in FIG. 2, the 3D image obtained from tissue-enhanced clearing (CLARITY) shows the abnormal vascular network and distribution of ultrasound-mediated drug-induced apoptosis cancer cell death in cholangiocarcinoma.

[0055] In particular, as confirmed in the images of Figures 2(b1) to 2(b3), in the absence of ultrasound irradiation (US), apoptotic cancer cells were observed mainly in the perivascular region of the control group through the coexistence of blood vessels and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) signals.

[0056] Also, FIGS. 2(a1) to FIGS. 2(a3) show a constant ISPTA of 0.5 W / cm² 2 This is the result of comparing groups treated with ultrasound at various duty cycles of 5%, 22%, and 45%. Fig. 2(a1) is a schematic diagram illustrating the process of low-intensity pulsed ultrasound (LIPUS) treatment on mice with HuCCT1 tumors treated with gemcitabine and cisplatin (Gem / Cis); Fig. 2(a2) is a photograph of a mouse receiving low-intensity pulsed ultrasound (LIPUS); and Fig. 2(a3) is a graph showing the change in duty cycle. Fig. 2(b1) is a 3D vascular image through blood vessel and TUNEL staining; Fig. 2(b2) is an enlarged image of the white square area in Fig. 2(b1); and Fig. 2(b3) is a reconstructed image thereof.

[0057] Figures 2(c1), 2(c2), 2(d1), 2(d2), 2(e1), and 2(e2) show the time elapsed after 20 minutes following drug injection, and it was found that an increase in the duty cycle was correlated with an increase in drug-induced apoptotic death.

[0058] And, FIGS. 2(c1) to FIGS. 2(h2) are TUNEL-stained 3D vascular images obtained from mice receiving low-intensity pulsed ultrasound (LIPUS) at duty cycles of 5%, 22%, and 45%, respectively, at the time of drug injection 20 minutes or 24 hours after drug injection. Specifically, FIGS. 2(c1) and 2(c2) show low-intensity pulsed ultrasound (LIPUS) treatment at a 5% duty cycle 20 minutes after drug infusion, FIGS. 2(d1) and 2(d2) show low-intensity pulsed ultrasound (LIPUS) treatment at a 22% duty cycle 20 minutes after drug infusion, FIGS. 2(e1) and 2(e2) show low-intensity pulsed ultrasound (LIPUS) treatment at a 45% duty cycle 20 minutes after drug infusion, FIGS. 2(f1) and 2(f2) show low-intensity pulsed ultrasound (LIPUS) treatment at a 5% duty cycle 24 hours after drug infusion, FIGS. 2(g1) and 2(g2) show low-intensity pulsed ultrasound (LIPUS) treatment at a 22% duty cycle 24 hours after drug infusion, and FIGS. 2(h1) and 2(h2) are Low-intensity pulsed ultrasound (LIPUS) was administered at a 45% duty cycle 24 hours after drug injection, and Figures 2(i) and 2(j) are representative 3D reconstructed images showing the infiltration gradient of apoptotic cancer cells into blood vessels at the 20-minute mark with a 5% duty cycle and the 24-hour mark with a 45% duty cycle, respectively.

[0059] When ultrasound was applied 24 hours after drug injection, as shown in Figs. 2(f), 2(g), and 2(h), it was confirmed that the apoptotic effect of ultrasound-mediated chemotherapy increased more dramatically than when treatment was administered 20 minutes after drug injection. In particular, severe apoptotic death was observed at a 45% duty cycle (24 hours after injection).

[0060] Figure 3 is a graph showing the number of cancer cells (vertical axis) that died from apoptosis induced by gemcitabine and cisplatin (Gem / Cis) treatment in combination with low-intensity pulsed ultrasound (LIPUS) irradiation under the same conditions as Figure 2 and under various duty cycles (5%, 22%, 45%) and treatment delays (20 minutes and 24 hours) relative to the distance from the blood vessel (horizontal axis). As shown in Figures 3(k1) and 3(k2), it can be confirmed that ultrasound irradiation is more effective than ultrasound non-irradiation (US), and that ultrasound irradiation is more effective as the duty cycle increases (e.g., 45%) and after 24 hours.

[0061] FIG. 4 shows ISPTA 0.1 W / cm² at 24 hours after drug injection according to the present invention. 2 , 0.3W / cm 2 , 0.5W / cm 2 , 0.7W / cm 2 This is an example of a 3D clear tumor image obtained from a cholangiocarcinoma (CCA) mouse that received low-intensity pulsed ultrasound (LIPUS) under the conditions (Fig. 4(a2)). Fig. 4(a1) is a schematic diagram illustrating the process of low-intensity pulsed ultrasound (LIPUS) treatment on mice with HuCCT1 tumors treated with gemcitabine and cisplatin (Gem / Cis), and Fig. 4(a2) shows changes in ultrasound ISPTA. Figs. 4(b1) to 4(e2) are TUNEL-stained 3D vascular images obtained from mice that received low-intensity pulsed ultrasound (LIPUS) under each ISPTA condition.

[0062] And, Fig. 5 shows various ISPTA (0.1W / cm²) as in Fig. 4 2 , 0.3W / cm 2 , 0.5W / cm 2 , 0.7W / cm 2This is a graph showing the number of apoptotic cancer cells induced by treatment with gemcitabine and cisplatin (Gem / Cis) combined with low-intensity pulsed ultrasound (LIPUS) in relation to the distance from blood vessels under ) conditions. Through Figures 4 and 5, ISPTA 0.5W / cm 2 and 0.7W / cm 2 It was observed that the highest number of apoptotic cancer cells occurred at [location]. Furthermore, it was confirmed that high ISPTA levels are associated with increased drug-induced apoptotic death. Quantitative analysis of apoptotic death as a function of distance from blood vessels revealed that the ultrasound-treated group showed a higher number of apoptotic cells at the same distance from blood vessels (see Fig. 5). ISPTA 0.7 W / cm² 2 0.5W / cm² under conditions 2 Compared to, the drug distribution was wider (at the same duty cycle) and was particularly more pronounced in regions far from blood vessels (>60 μm). However, the total number of apoptosis remained similar between the two conditions (see Fig. 5).

[0063] Figure 6 is an example of 3D clear tumor images (Figs. 6(b1), 6(b2), 6(b3)) obtained from mice with cholangiocarcinoma (CCA) irradiated with low-intensity pulsed ultrasound (LIPUS) and 3D clear tumor images (Figs. 6(c1) to 6(f3)) obtained from mice with cholangiocarcinoma (CCA) irradiated with ultrasound at duty cycles of 5%, 22%, and 45% (Figs. 6(a1) to 6(a3)) 24 hours after injection of liposome nanoparticles (Lip@Ru) according to the present invention.

[0064] Figures 6(a1) and 6(a2) are schematic diagrams illustrating the process of low-intensity pulsed ultrasound (LIPUS) treatment on mice with HuCCT1 tumors treated with liposome nanoparticles (Lip@Ru), and Figure 6(a3) includes variations in the ultrasound duty cycle. Figures 6(b1) to 6(e3) are TUNEL-stained 3D vascular images obtained from mice that received low-intensity pulsed ultrasound (LIPUS) under respective duty cycle conditions, and Figures 6(f1) to 6(f3) show ISPTA 0.7 W / cm² 2 This is a TUNEL-stained 3D vascular image obtained from a mouse that received low-intensity pulsed ultrasound (LIPUS) under conditions of a 45% duty cycle.

[0065] And Figure 7 is a graph showing the number of cancer cells that absorbed ruthenium (Ru) by low-intensity pulsed ultrasound (LIPUS) irradiation in relation to the distance from the blood vessel under various duty cycles (5%, 22%, 45%), as in Figure 6.

[0066] As can be seen in Figures 6 and 7, Figures 6(b1) through 6(f1) show that the distribution of ruthenium increases at high duty cycles when ultrasound is applied. Furthermore, Figure 7 clearly indicates that the number of cancer cells absorbing ruthenium (Ru) increases at high duty cycles at the same distance from the blood vessel. Duty cycle 45% and power 0.7 W / cm² 2 At, 45% and power 0.5W / cm² 2 In comparison, increased uptake of ruthenium (Ru) by cancer cells was observed in regions far from blood vessels (>60 μm). However, ISPTA was 0.5 W / cm 2 When it exceeded , the number of cancer cells that absorbed ruthenium (Ru) became saturated.

[0067] FIG. 8 shows ISPTA 0.1 W / cm² at 24 hours after injection of liposome nanoparticles (Lip@Ru) according to the present invention. 2 , 0.3W / cm 2 , 0.5W / cm 2, 0.7W / cm 2 This is an example of a 3D clear tumor image obtained from a cholangiocarcinoma (CCA) mouse that received low-intensity pulsed ultrasound (LIPUS) under the conditions shown. Specifically, Figures 8(a1) and 8(a2) are schematic diagrams illustrating the process of treating mice with HuCCT1 tumors treated with liposomal nanoparticles (Lip@Ru) with low-intensity pulsed ultrasound (LIPUS), and Figure 8(a3) is a graph showing the change in ISPTA. Figures 8(b1) to 8(e2) are TUNEL-stained 3D vascular images obtained from mice that received low-intensity pulsed ultrasound (LIPUS) under each ISPTA condition. And, Figure 9 is a graph showing the number of cancer cells that absorbed liposomal nanoparticles (Lip@Ru) according to the distance from the blood vessel under the same conditions as Figure 8.

[0068] As shown in Figures 8 and 9, in the control group without ultrasound treatment, the liposomal nanoparticles (Lip@Ru) were mainly distributed inside and near the blood vessels. However, upon ultrasound treatment, as the ISPTA level increased, a more pronounced diffusion of nanoparticles outward from the blood vessels was observed. As ISPTA increased, more ruthenium (Ru)-absorbing cancer cells were observed at the same distance from the blood vessels, and the maximum distance increased further (see Figure 9). However, the ultrasound-induced nanoparticle diffusion effect was observed at an ISPTA of 0.5 W / cm² 2 Even when exceeded, it did not result in an additional increase in the number of cancer cells that absorbed ruthenium (Ru).

[0069] FIG. 10(a) is a schematic diagram showing the process of drug movement into a hypoxic region by unidirectional flow and sonoporation induced by low-intensity pulsed ultrasound (LIPUS) of the present invention, and FIG. 10(b) is a graph showing the ratio of Hif-1α-expressing cells and apoptotic Hif-1α cells in relation to the distance from blood vessels. In addition, under the same conditions, FIG. 11 is an example of a 3D cleared tumor image showing the spatial distribution of apoptotic cancer cells in a hypoxic region and under hypoxic conditions in cholangiocarcinoma (CCA) after tissue clearing according to the present invention. More specifically, FIG. 11(c) shows an image of the cleared tumor tissue immunostained with Hif-1α cells (purple), blood vessels (red), apoptosis (green), and nuclei (blue), respectively. Among these, Fig. 11(d) is an image of blood vessels (red), Fig. 11(e) is an image of Hif-1α cells (purple), Fig. 11(f) is an image of apoptotic cells (TUNEL green), and Fig. 11(g) is an image showing both Hif-1α cells and blood vessels. Additionally, Fig. 11(h1) shows Hif-1α cells and apoptotic cells along with blood vessels, and Fig. 11(h2) is an enlarged image of Fig. 11(h1). In particular, the white arrow in Fig. 11(h2) indicates apoptotic Hif-1α cells.

[0070] Fig. 11(i) is a 3D reconstructed image showing Hif-1α-expressing cells (purple dots) and apoptotic cancer cells (green dots) along with a 3D vascular structure (red), Fig. 11(j1) is a 3D image showing that the color changes from blue to red as the coexistence distance of Hif-1α-expressing cells (purple) and apoptotic cancer cells (green), indicated by spectral dots at relative distance from the blood vessels, increases, and Fig. 11(j2) is an enlarged image of Fig. 11(j1).

[0071] As shown in Figs. 10 and 11, the anticancer agent was efficiently transported into the hypoxic zone via ultrasound-induced unidirectional flow (ISPTA 0.5 W / cm² and DC 45%). Expression of Hif-1α (Hypoxia-Inducible Factor 1-alpha) protein was strongly detected in regions far from blood vessels (see Figs. 11(e) and 11(g)), and apoptotic death was observed in distant regions outside the blood vessels, including blood vessels (see Figs. 11(d) and 11(f)).

[0072] As shown in Figures 11(h1) and 11(h2), the coexistence of Hif-1α expression and TUNEL signaling confirmed that drug penetration from blood vessels to deep hypoxic cancer cells was effectively achieved by ultrasound. Through the 3D image reconstruction process, Hif-1α-positive cancer cells were indicated as purple dots, and apoptotic cancer cells as green dots. As plotted in Figure 10, Hif-1α-positive cancer cells were distributed from approximately 30 μm to 310 μm from the blood vessels. Cancer cells expressing Hif-1α and undergoing apoptosis were selectively identified as coexisting using software (see Experimental Section), and their distance from the blood vessels was calculated. The distance from the blood vessels to the Hif-1α-positive apoptotic cancer cells was calculated to be approximately 190 μm (see Figures 11(j1) and 11(j2)).

[0073] FIGS. 12 to 15 are graphs showing the results of a statistical analysis of low-intensity pulsed ultrasound (LIPUS)-mediated induced cell death and Lip@Ru uptake of gemcitabine and cisplatin (Gem / Cis) under various duty cycles and ISPTA according to the present invention. Specifically, FIG. 12(a) shows ISPTA 0.5 W / cm² under various duty cycles. 2 This is a graph showing the number of apoptotic cancer cells after administering gemcitabine and cisplatin (Gem / Cis) along with low-intensity pulsed ultrasound (LIPUS) under conditions.

[0074] Fig. 12(b) is a graph showing the number of apoptotic cancer cells after administration of gemcitabine and cisplatin (Gem / Cis) along with low-intensity pulsed ultrasound (LIPUS) under various ISPTA conditions at a duty cycle of 22%, Fig. 12(c) is a graph showing the total number of cancer cells that absorbed liposomal nanoparticles (Lip@Ru) under ISPTA conditions of 0.5 W / cm² under various duty cycles, and Fig. 12(d) is a graph showing the total number of cancer cells that absorbed liposomal nanoparticles (Lip@Ru) at various ISPTA levels under a duty cycle of 22%.

[0075] Figure 13(e) is a comparison graph of the cell death efficiency of cancer cells when ultrasound is applied after 20 minutes and after 24 hours under each duty cycle condition, Figure 13(f) is a comparison graph of the cell death efficiency of cancer cells under a 5% duty cycle condition, Figure 13(g) is a comparison graph under a 22% condition, and Figure 13(h) is a comparison graph under a 45% duty cycle condition.

[0076] FIG. 14(i) shows ISPTA 0.5 W / cm under various duty cycle conditions. 2 Figure 14(j) is a graph of the number of cancer cells induced to apoptosis by gemcitabine and cisplatin (Gem / Cis) at distances of 60 μm, 100 μm, and 150 μm or more from blood vessels under a duty cycle of 22% and the number of cancer cells induced to apoptosis by gemcitabine and cisplatin (Gem / Cis) at various ISPTA levels at distances of 60 μm, 100 μm, and 150 μm or more from blood vessels under a duty cycle of 22%.

[0077] Fig. 14(k) shows ISPTA 0.5 W / cm² under various duty cycle conditions. 2Figure 14(l) is a graph of the number of cancer cells that absorbed liposome nanoparticles (Lip@Ru) at distances of 60 μm, 100 μm, and 150 μm or more from blood vessels under a duty cycle of 22% and various ISPTA levels at distances of 60 μm, 100 μm, and 150 μm or more from blood vessels.

[0078] Fig. 15(m) is a 3D graph showing the distribution of gemcitabine and cisplatin (Gem / Cis)-induced apoptosis at a distance of 60 μm or more from blood vessels, considering ISPTA and duty cycles, and Fig. 15(n) is a 3D graph showing the distribution of cancer cells absorbing liposome nanoparticles (Lip@Ru) at a distance of 60 μm or more from blood vessels, considering ISPTA and duty cycles.

[0079] As shown in Figs. 12 to 15, when ultrasound treatment was performed 24 hours after administration of gemcitabine and cisplatin (Gem / Cis), the total number of apoptotic cancer cells increased 2.13-fold at a 5% duty cycle, 4.15-fold at a 22% duty cycle (p<0.01), and 5.53-fold at a 45% duty cycle (p<0.01) (see Fig. 12(a)). Similarly, when ultrasound treatment was performed 24 hours after administration of gemcitabine and cisplatin (Gem / Cis), ISPTA 0.1 W / cm² 2 At total apoptosis, cancer cells were 1.58 times higher, 0.3 W / cm² 2 It increased 2.21-fold at 0.5 W / cm², 4.14-fold at 0.5 W / cm² (p<0.05), and 4.21-fold at 0.7 W / cm² (p<0.01) (see Fig. 12(b)). The same ISPTA 0.5 W / cm² 2The total number of cancer cells that absorbed ruthenium (Ru) increased 1.75-fold at a duty cycle of 5%, 4.37-fold at 22% (p<0.01), and 5.49-fold at 45% (p<0.001) (see Fig. 12(c)). Similarly, the total number of cancer cells that absorbed ruthenium (Ru) at a duty cycle of 22% was ISPTA 0.1 W / cm² 2 1.4 times at, 0.3 W / cm² 2 2.42 times at 0.5 W / cm², 4.37 times at 0.7 W / cm² (p<0.01) 2 It increased 4.12 times (p<0.05).

[0080] In addition, to determine the clinically optimal timing for ultrasound irradiation, the efficacy of drug-induced apoptosis was compared when ultrasound was applied 20 minutes or 24 hours after drug administration. At duty cycles of 5%, 22%, and 45%, the efficacy increased by 1.5-fold, 3.77-fold (p<0.05), and 4.6-fold (p<0.05) after 24 hours, respectively. Furthermore, at the same duty cycle of 45%, the total number of cancer cells absorbing ruthenium (Ru) at ISPTA 0.5 W / cm² and 0.7 W / cm² was 43,014 and 42,975, respectively, with no significant difference (see Fig. 13(h)).

[0081] At duty cycles of 5%, 22%, and 45%, under ISPTA conditions of 0.5 W / cm², the number of gemcitabine and cisplatin (Gem / Cis)-induced apoptotic cancer cells located at a distance of 60 μm or more increased by 8.62-, 12.7-, and 18.5-fold, respectively, compared to the control group (see Fig. 14(i)). At a distance of 60 μm or more, the number of apoptotic cancer cells was 214 at a duty cycle of 0%, 1,845 at a duty cycle of 5%, 2,730 at a duty cycle of 22%, and 3,962 at a duty cycle of 45% (see Fig. 14(i)). In addition, at a distance of 100 μm or more, the number of apoptotic cancer cells was 6 at a duty cycle of 0%, 422 at a duty cycle of 5%, 450 at a duty cycle of 22%, and 1,079 at a duty cycle of 45% (see Fig. 14(i)). Furthermore, at a distance of 150 μm or more, the number of gemcitabine and cisplatin (Gem / Cis)-induced apoptotic cancer cells was 0, 16, 25, and 74, respectively (see Fig. 14(i)).

[0082] ISPTA 0.1W / cm 2 , 0.3W / cm 2 , 0.5W / cm 2 , 0.7W / cm 2 When ultrasound was applied at a duty cycle of 22%, the number of apoptotic cancer cells at a distance of 60 μm or more was compared to the control group (0 W / cm²). 2 Compared to ), the numbers increased by 6.11, 10, 11.1, and 19.8 times, respectively (see Fig. 14(j)). At a distance of 60 μm or more, the number of apoptotic cancer cells was 214 at ISPTA 0 and at ISPTA 0.1 W / cm², respectively. 2 1,308 units, ISPTA 0.3W / cm² 2 2,141 units, ISPTA 0.5W / cm² 2 2,384 units, ISPTA 0.7W / cm² 2It was found to be 4,239 (see Fig. 14(j)). In addition, at a distance of 100 μm or more, the number of apoptotic cancer cells was 6 at ISPTA 0 and 0.1 W / cm², respectively. 2 301 units, ISPTA 0.3W / cm² 2 545 units at, ISPTA 0.5W / cm² 2 450 units, ISPTA 0.7W / cm² 2 It was found to be 1,468 (see Fig. 14(j)). In addition, the number of cancer cells subjected to gemcitabine and cisplatin (Gem / Cis)-induced apoptosis at a distance of 150 μm or more was found to be 0, 0, 27, 101, and 374, respectively (see Fig. 14(j)).

[0083] The number of cancer cells that absorbed liposome nanoparticles (Lip@Ru) was at ISPTA 0.5W / cm² at duty cycles of 5%, 22%, and 45% 2 Under the conditions, at a distance of 60 μm or more, the number of cancer cells increased by 3.67-fold, 10.81-fold, and 15.5-fold, respectively, compared to the control group (duty cycle 0%) (see Fig. 14(k)). The number of cancer cells that absorbed Lip@Ru at a distance of 60 μm or more was 374 at duty cycle 0%, 1,373 at duty cycle 5%, 4,045 at duty cycle 22%, and 5,830 at duty cycle 45%, respectively (see Fig. 14(k)). Additionally, the number of cancer cells that absorbed liposome nanoparticles (Lip@Ru) at a distance of 100 μm or more was 24 at duty cycle 0%, 350 at duty cycle 5%, 557 at duty cycle 22%, and 1,086 at duty cycle 45%, respectively (see Fig. 14(k)). In addition, the number of cancer cells that absorbed liposome nanoparticles (Lip@Ru) at a distance of 150 μm or more was 0 at a duty cycle of 0%, 23 at a duty cycle of 5%, 107 at a duty cycle of 22%, and 114 at a duty cycle of 45% (see Fig. 14(k)).

[0084] ISPTA 0W / cm 2 , 0.1W / cm2 , 0.3W / cm 2 , 0.5W / cm 2 , 0.7W / cm 2 When ultrasound was treated with a 22% duty cycle, the number of apoptotic cancer cells at a distance of 60 μm or more was, respectively, compared to the control group (ISPTA 0W / cm²). 2 Compared to ), the number of apoptotic cancer cells increased by 3.91, 5.52, 10.81, and 12.85 times (see Fig. 14(l)). At a distance of 60 μm or more, the number of apoptotic cancer cells was 374 at ISPTA 0 and at ISPTA 0.1 W / cm², respectively. 2 1,464 units at ISPTA 0.3W / cm², 2,065 units at ISPTA 0.5W / cm² 2 4,045 units, ISPTA 0.7W / cm² 2 It was found to be 4,809 (see Fig. 14(l)). In addition, at a distance of 100 μm or more, the number of apoptotic cancer cells was 24 at ISPTA 0 and at ISPTA 0.1 W / cm², respectively. 2 258 units at, ISPTA 0.3W / cm² 2 282 units at, ISPTA 0.5W / cm² 2 557 units at, ISPTA 0.7W / cm² 2 It was found to be 868 (see Fig. 14(l)). In addition, at a distance of 150 μm or more, the number of cancer cells subjected to gemcitabine and cisplatin (Gem / Cis)-induced apoptosis was 0 at ISPTA 0 and 0 at ISPTA 0.1 W / cm², respectively. 2 17 at, ISPTA 0.3W / cm 2 7 units, ISPTA 0.5W / cm² 2 107 units, ISPTA 0.7W / cm² 2 It was found to be 26 (see Fig. 14(l)).

[0085] Figure 15(m) is a 3D graph showing the distribution of apoptotic cancer cells at a distance of more than 60 μm from blood vessels, considering ISPTA and the duty cycle. Drug delivery and cell death within the hypoxic region improved as the duty cycle or ISPTA increased. The most favorable conditions for drug-induced cell death under hypoxic conditions were an ISPTA of 0.7 W / cm². 2 -DC 22% and ISPTA 0.5W / cm² 2 It was found to be -DC 45%. Figure 15(n) is a 3D graph showing the distribution of cancer cells absorbing liposome nanoparticles (Lip@Ru) at a distance of more than 60 μm from blood vessels, considering ISPTA and the duty cycle. The nanoparticle delivery effect under hypoxic conditions improved as the duty cycle or ISPTA increased. The condition in which the nanoparticle delivery effect under hypoxic conditions was most favorable was ISPTA 0.5 W / cm². 2 DC was 45%.

[0086] FIGS. 16 and 17 are a schematic diagram and a graph illustrating tumor growth profiles resulting from combination therapy with an anticancer agent and low-intensity pulsed ultrasound (LIPUS) in cholangiocarcinoma (CCA) according to the present invention. Specifically, in FIG. 16, Group 1 is a control group in which physiological saline was injected into mice with tumors; Group 2 received monotherapy with gemcitabine and cisplatin (Gem / Cis); and Groups 3, 4, and 5 received low-intensity pulsed ultrasound (LIPUS) therapy (ISPTA 0.5 W / cm²) simultaneously with gemcitabine and cisplatin (Gem / Cis) therapy, respectively. 2 , DC 22%, ISPTA 0.7W / cm 2 , DC 45%, ISPTA 0.5W / cm 2 This is a group of mice with tumors that received [DC 45%). Mice were injected with gemcitabine and cisplatin (Gem / Cis) on days 1 and 8, and low-intensity pulsed ultrasound (LIPUS) was performed 24 hours later. Figure 17 is a graph showing the tumor growth size monitored every two days for 21 days from this group.

[0087] As shown in Figures 16 and 17, the antitumor efficacy of in vivo ultrasound-mediated drug therapy was evaluated using five groups. Group 1 was a control group in which physiological saline was injected into mice with tumors, and Group 2 received gemcitabine and cisplatin (Gem / Cis) monotherapy. Groups 3, 4, and 5 received ultrasound therapy (ISPTA 0.5 W / cm²) simultaneously with gemcitabine and cisplatin (Gem / Cis) treatment, respectively. 2 , DC 22%, ISPTA 0.7W / cm 2 , DC 45%, ISPTA 0.5W / cm 2 Mice with tumors were divided into groups that received DC (45%). Compared to Group 1, tumor growth in Group 2 was reduced due to the efficacy of the anticancer drug. In particular, in Groups 3, 4, and 5, ultrasound therapy exerted a synergistic effect with drug treatment, clearly showing controlled modulation of tumor growth rates compared to Group 2. Notably, Group 5 showed the most prominent effect in inhibiting tumor growth, while there was no significant difference between Groups 4 and 5. Through these results, it can be confirmed that the tumor size of Groups 3, 4, and 5 did not increase significantly, whereas the tumor size of Groups 1 and 2 increased significantly.

[0088] Experimental Example

[0089] cell culture

[0090] The cholangiocarcinoma (CCA) cell line HuCCT1 was purchased from the RIKEN BRC Cell Bank in Japan. This cell line was cultured in RPMI 1640 medium containing 10% fetal calf serum and 1% penicillin-streptomycin at 37°C in a 5% CO2 environment.

[0091] Transplantable mouse model

[0092] All animal experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University. Seven-week-old female BALB / c nude mice (approx. 20 g) were used in this study and were purchased from Orient Bio Inc. in Korea. For tumor transplantation, 4 × 10⁶ HuCCT1 cells were used. 6 The mixture was mixed with 100 μL of Geltrex and injected subcutaneously into the right flank of each mouse.

[0093] Effect of ultrasound on drug penetration of nanoparticles in the tumor microenvironment (TME)

[0094] The tumor size is approximately 1,000 mm 3 Upon reaching [the target], experiments to determine the distribution of nanoparticles were initiated. Ruthenium (Ru)-encapsulated liposomes (Lip-Ru: 8 mg / kg) were intravenously injected into HuCCT1-transplanted mice. Twenty-four hours after the intravenous injection, the tumor site was exposed to ultrasound for one hour (pulsed ultrasound operating at a fundamental frequency of 250 kHz). The tumor was excised after 48 hours. Immediately before tumor excision, tomato lectin (0.1 mg) labeled DyLight 649 was administered via tail vein injection. Ten minutes later, the mice were euthanized by CO2 asphyxiation. The tumors were washed with 1×PBS and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. Untreated mice (HuCCT1-transplanted mice injected with Lip-Ru) were used as a negative control.

[0095] Intratumoral ultrasound for cisplatin and gemcitabine treatment

[0096] Cisplatin and gemcitabine were intravenously administered to HuCCT1-transplanted mice as follows: gemcitabine 200 mg / kg, diluted in 0.1 ml of normal saline, administered intravenously; cisplatin 5 mg / kg, diluted in 0.1 ml of normal saline, administered intravenously. Pulsed ultrasound was applied to the HuCCT1 tumors for 1 hour, 20 minutes or 24 hours after intravenous administration. The doses of gem / cis used in the mice were determined via human-animal dose conversion based on doses used in clinical patients. Tumors were excised after 48 hours. Immediately before tumor excision, tomato lectin (0.1 mg) labeled DyLight 649 was administered via tail intravenous injection. Ten minutes later, the mice were euthanized by CO2 asphyxiation. The tumors were washed with 1×PBS and fixed overnight in 4% paraformaldehyde (PFA) at 4°C.

[0097] Organizational transparency

[0098] PFA-fixed tumors were sliced ​​to a thickness of 1 mm and immersed in X-CLARITY Hydrogel Solution at 4°C for 24 hours to ensure uniform diffusion of hydrogel monomers throughout the sample. Subsequently, oxygen was removed from the tube using nitrogen gas for approximately 3 minutes. The tumor samples were polymerized in the tubes in a 37°C water bath for 3–4 hours. Then, an active clearing process was performed using the X-CLARITY tissue clearing system. This was conducted at 37°C using a 0.9A current, and the system accelerated the process of removing lipids from the tissue through solution circulation. A tissue clearing solution optimized for the system (Logos Biosystems) and a Sodium Dodecyl Sulfate (SDS)-based solution with a pH of 8.5 were used. The cleared tumor tissue was rinsed with 1×PBS at 37°C for 2 days.

[0099] Cell death induced by cisplatin / gemcitabine in HuCCT1 grafts

[0100] To detect apoptosis induction in the tumor microenvironment (TME), cleared 1 mm thick tumor tissues were stained using the TUNEL staining kit. First, the tumor tissues were incubated in permeation buffer at 4°C for 4 hours. Then, the tumor tissues were incubated in a staining solution (TdT enzyme + Hoechst 33342 + labeling safety buffer) in a tabletop incubator at 37°C overnight and washed with 1×PBS at 37°C. Finally, the stained samples were rinsed in DDW for 10 minutes (repeated twice) and subsequently immersed in X-CLARITY™ mounting solution (RI=1.46, Logos Biosystems) at room temperature.

[0101] 3D tissue imaging of Lip-Ru distribution in HuCCT1 grafts

[0102] Tumor tissue immersed in the mounting solution was sealed in a cover glass and a bottom confocal dish. The tumor tissue was imaged using a Leica TCS SP8 DMI8-CS system, which was equipped with an HC PL APO CS 10x / 0.40 DRY objective (Leica Microsystems GmbH Wetzlar, Germany). 3D fluorescence images were acquired using the Z-stacking function (Hoechst 33342: 405 nm, Lip-Ru: 488 nm, Vascular: 633 nm excitation). The X / Y resolution was set to 1024×1024 pixels (scan depth 350 μm).

[0103] 3D tissue imaging of cisplatin / gemcitabine-induced cell death distribution in HuCCT1 grafts

[0104] TUNEL-stained tumor tissues were immersed in X-CLARITY™ mounting solution (RI=1.46, Logos Biosystems) at room temperature and imaged using a Leica TCS SP8 DMI8-CS system. The system was equipped with HC PL APO CS 10x / 0.40 DRY objective. 3D fluorescence images were acquired using the Z-stacking function (Hoechst 33342: 405 nm, cell death: 488 nm, blood vessels: 633 nm excitation). The X / Y resolution was set to 1024×1024 pixels (scan depth 350 μm).

[0105] Image analysis of Lip-Ru distribution in HuCCT1 grafts

[0106] 3D images were evaluated using the Bitplane Imaris CL and XT software packages. In the Imaris software, the 'spots' application was initially used to determine the number of nuclei. Channel 1 (blue) was selected to isolate a specific region of interest (ROI) during the algorithm phase of the spots application. The estimated diameter for detection was set to 5 μm, a value determined using the measurement point application in the slice view. Next, the quality filter value was manually adjusted to be greater than 10 in the lower threshold control box. Then, the 'surface' application was used for blood vessel segmentation. The region of interest (ROI) was set to cover the entire image, and Channel 2 (blood vessel - red) was selected as the source channel. The blood vessels were segmented by applying smoothing using the optimal filter value and manually setting the threshold. Subsequently, the 'surface' application was used for segmentation of the Lip-Ru bound cancer cell membrane. Again, the region of interest (ROI) was set to cover the entire image, and Channel 3 (Lip-Ru: green channel) was selected as the source channel. The Lip-Ru signal was smoothed with optimal filter values ​​and segmented using manually set thresholds. Finally, nuclei near the Lip-Ru surface were identified using the 'find spots close to surface' application, and 3D Euclidean distance transformations were automatically performed near the Lip-Ru surface outside the blood vessels. The drug penetration (escape distance) of Lip-Ru from intratumoral blood vessels within the tumor microenvironment (TME) was measured in micrometers. Based on 3D reconstructed images under all conditions, the number of cancer cells absorbing Lip-Ru as a function of distance from blood vessels was graphed. Three sets of tumor tissues were analyzed, and the average values ​​were graphed.

[0107] Image analysis of the distribution of cisplatin / gemcitabine-induced cell death in HuCCT1 grafts

[0108] Distance maps were constructed by generating 3D reconstructed images to evaluate the distribution of cell death in relation to blood vessels. As previously mentioned, the 'spot' and 'surface' applications were used to determine the distribution of cell death within the tumor microenvironment (TME). Briefly, nuclei were first quantitatively determined using the 'spots' application. Channel 1—blue (nuclear signal)—was selected with the 'Segment specific Region of Interest (ROI) only' option during the image processing step of the spot application, and then the entire image was processed. The 'Estimated Diameter' was set to 5 μm. Next, the quality filter value was manually set to 5 or higher in the lower threshold control box. Second, the 'surface' application was used for blood vessel segmentation. The region of interest (ROI) was set to the entire image, and the source channel was selected as Channel 2 (red signal). The blood vessel signal was smoothed using the optimal filter value and then segmented through a manually selected threshold (background removal). Third, the 'spot' application was used to determine the amount of nuclei containing the cell death signal (Channel 3—green signal). The spot detection 'estimated diameter' was set to 5 μm. The 5 μm value for the nucleus can be determined using the measurement point application in the slice view. Then, the quality filter value was manually set to 5 or higher in the lower threshold control box. Finally, nuclei surrounded by spots (channel 1 - blue signal) near the cell death spot (channel 3 - green signal) were identified via the 'find spots close to spots' application, and 3D Euclidean distance transformations were automatically performed outside the spots near the blood vessels. Cell death induced by cisplatin / gemcitabine was measured in micrometers within the tumor microenvironment (TME). The distance of cell death from intratumoral blood vessels was also obtained.Based on 3D reconstructed images under all conditions, the number of dead cancer cells according to distance from blood vessels was graphed. Three sets of tumor tissues were analyzed, and the average values ​​were graphed.

[0109] In vivo efficacy study

[0110] Experiments to evaluate the antitumor efficacy (tumor growth inhibition) of ultrasound in cisplatin and gemcitabine treatments were conducted on tumor volumes of 150-200 mm 2 It started when it reached. Tumor volume was measured every two days using a caliper, and the calculated volume was calculated using the following formula: V=(W 2 ×L) / 2 (W and L represent the width and length of the tumor). Tumors 150 ~ 200 mm 3 Upon reaching the required volume, the mice were randomly divided into five groups of 20: Group 1: Control group, Group 2: Gemcitabine and cisplatin group, Group 3: Group treated with ultrasound along with gemcitabine and cisplatin (ISPTA 0.5W / cm² 2 DC 22%), Group 4: Group treated with ultrasound together with gemcitabine and cisplatin (ISPTA 0.7W / cm²) 2 DC 45%), Group 5: Group treated with ultrasound with gemcitabine and cisplatin (ISPTA 0.5W / cm² 2 DC 45%). Cisplatin (5 mg / kg) and gemcitabine (200 mg / kg) were intravenously administered to mice on days 1 and 8, respectively. Doses were determined based on previous literature and pharmaceutical company recommendations, referencing the FDA-guided formula for converting human doses to animal doses. Ultrasound was applied on days 2 and 9, 24 hours after drug treatment. Tumor size was measured every two days, and tumor sizes were compared among the four groups.

[0111] As described above, the detailed description of the preferred embodiments of the present invention disclosed is provided to enable those skilled in the art to implement and practice the present invention. Although the present invention has been described with reference to preferred embodiments, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the invention. For example, those skilled in the art may utilize each configuration described in the embodiments described above in combination with one another. Accordingly, the present invention is not intended to be limited to the embodiments shown herein, but to be given the broadest scope consistent with the principles and novel features disclosed herein.

[0112] The present invention may be embodied in other specific forms without departing from the spirit and essential features of the invention. Accordingly, the above detailed description should not be interpreted restrictively in all respects but should be considered exemplary. The scope of the invention shall be determined by a reasonable interpretation of the appended claims, and all modifications within the equivalent scope of the invention are included within the scope of the invention. The invention is not intended to be limited to the embodiments shown herein, but to be given the broadest possible scope consistent with the principles and novel features disclosed herein. Furthermore, embodiments may be constructed by combining claims that are not explicitly related in the claims, or by including them as new claims through amendments made after filing.

Claims

1. A step of administering a drug and a fluorescent substance to cholangiocarcinoma (CCA) under a tumor microenvironment (S100); Step (S120) in which a predetermined time elapses; Step (S140) of irradiating the cholangiocarcinoma (CCA) with low-intensity pulsed ultrasound (LIPUS); A step (S160) of obtaining a 3D image by performing tissue enhancement clarity on the above-mentioned cholangiocarcinoma (CCA); A step of confirming the drug permeability of the drug and the fluorescent substance in vivo from the above 3D image (S180); and A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized by including the step (S200) of confirming the distribution of apoptosis within the cholangiocarcinoma (CCA) from the above 3D image, and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

2. In Paragraph 1, A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) under a tumor microenvironment characterized in that the above-mentioned cholangiocarcinoma (CCA) is hypoxic cholangiocarcinoma (CCA), and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

3. In Paragraph 1, The above low-intensity pulsed ultrasound (LIPUS) has an ISPTA intensity of 0.1 to 0.7 W / cm² 2 A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment characterized by a duty cycle of 5 to 45%, and combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting cholangiocarcinoma growth.

4. In Paragraph 3, The ISPTA intensity of the above low-intensity pulsed ultrasound (LIPUS) is 0.5 W / cm² 2 A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized by a duty cycle of 45%, and combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting cholangiocarcinoma growth.

5. In Paragraph 3, The ISPTA intensity of the above low-intensity pulsed ultrasound (LIPUS) is 0.7 W / cm² 2 A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized by a duty cycle of 22%, and combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting cholangiocarcinoma growth.

6. In Paragraph 1, A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) under a tumor microenvironment, characterized in that the above-mentioned predetermined time is 24 hours, and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

7. In Paragraph 1, A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized in that the anticancer agent is gemcitabine and / or cisplatin, and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

8. In Paragraph 1, A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized in that the fluorescent material is fluorescent ruthenium and liposomal nanoparticles encapsulating the ruthenium, and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

9. In Paragraph 8, A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized in that the liposome nanoparticles are fluorescent ruthenium-liposome complexes, and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

10. In Paragraph 1, A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized in that in the step (S180) of confirming drug permeability, the drug permeability is calculated and confirmed based on pixel information regarding the penetration depth and distribution of the drug and the fluorescent substance, and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

11. In Paragraph 7, A method for confirming drug permeability and cancer cell apoptosis of cholangiocarcinoma by irradiation with low-intensity pulsed ultrasound (LIPUS) in a tumor microenvironment, characterized by inhibiting the growth of cholangiocarcinoma (CCA) due to the administration of the above gemcitabine and cisplatin and irradiation with the above low-intensity pulsed ultrasound (LIPUS), and a combination therapy of gemcitabine and cisplatin with low-intensity pulsed ultrasound for inhibiting the growth of cholangiocarcinoma.

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