KR102742323B1 - Tumor Targeting Exosome loaded with sonosensitizers and a preparing method thereof - Google Patents

Abstract

The present invention relates to an exosome having an enhanced tumor-targeting ability by loading an ultrasound sensitizer and conjugating a tumor-targeting substance, and a method for producing the same. The exosome according to the present invention accumulates specifically in tumor cells, does not cause in vivo toxicity due to the exosome itself, and can effectively act on tumors located deep in the body that cannot be treated with photodynamic therapy, and thus can be utilized as a non-invasive or minimally invasive treatment method for the treatment of various solid tumors.

Description

Tumor Targeting Exosome Loaded with Sonosensitizers and a Preparing Method thereof {Tumor Targeting Exosome Loaded with Sonosensitizers and a Preparing Method thereof}

The present invention relates to a tumor-targeting exosome loaded with an ultrasound sensitizer and a method for producing the same, and more particularly, to an exosome loaded with at least one ultrasound sensitizer selected from the group consisting of porphyrins, phthalocyanine compounds, rhodamine compounds, naphthalocyanines, BODIPY compounds, and indocyanine green and conjugated with a tumor-targeting agent to enhance tumor-targeting ability, and a method for producing the same.

Reactive oxygen species (ROS) are byproducts of cellular metabolic reactions in living organisms and are involved in cell signaling and homeostasis. As ROS have been found to kill cancer cells, novel anticancer therapies that control intracellular ROS have emerged. Photodynamic therapy (PDT) is a minimally invasive treatment method that uses exogenous ROS generated by a photosensitizer under light irradiation. Although PDT has low systemic toxicity and can selectively destroy tumors, its clinical application is limited due to the depth at which light can penetrate into tissues. Therefore, interest is focused on novel therapeutic systems that can generate ROS in deep tissues, and sonodynamic therapy (SDT) has attracted great attention as a potential alternative to PDT in cancer treatment because it utilizes ROS generated from an ultrasound sensitizer when exposed to ultrasound (US). Since ultrasound can penetrate deeper into tissues, SDT has the advantage of being applicable to the treatment of deep tumors that cannot be effectively treated with PDT. Additionally, SDT has the advantage of being able to selectively destroy cancer cells while minimizing damage to surrounding normal cells because the ultrasound can be precisely focused on a specific tumor area.

Since the therapeutic effect of SDT is dependent on the effect of ultrasound sensitizers, the development of safe and effective ultrasound sensitizers is particularly important for the success of SDT. Most organic ultrasound sensitizers are hydrophobic and thus tend to aggregate in the physiological environment. In addition, they have poor pharmacokinetic properties and low cancer specificity. To overcome these limitations of ultrasound sensitizers, nanocarriers have been recently developed (Maeda, H. et al., J. Controlled Release 2000, 65, 271-284). Nanocarriers can significantly improve the intracellular delivery of poorly soluble ultrasound sensitizers and preferentially transport ultrasound sensitizers to tumor areas through the enhanced permeability and retention (EPR) effect. However, some synthetic nanocarriers can induce unexpected toxicity and immunogenicity, so the development of safe and biocompatible nanocarriers encapsulating ultrasound sensitizers is very important for the clinical application of SDT.

Exosomes, nanosized (30-150 nm) membrane vesicles produced by cells, have recently emerged as versatile biocompatible nanocarriers. Since exosomes are of endogenous origin, they have excellent biocompatibility, long blood circulation half-life, and almost no immunogenicity. Therefore, exosomes have been considered as clinically applicable drug carriers that can efficiently transport various hydrophobic and hydrophilic drugs. In addition, exosomes have the advantage of selective accumulation in tumors through the EPR effect (Antimisiaris, S. et al., Pharmaceutics, 2018, 10, 218).

The present inventors have made great efforts to invent a method for delivering an ultrasound sensitizer to cancer cells specifically for effective SDT. As a result, they selected indocyanine green (ICG), a biocompatible near-infrared (NIR) absorbent approved by the Ministry of Food and Drug Safety, as an ultrasound sensitizer, and were able to simply manufacture an ultrasound sensitizer carrier in which indocyanine green is loaded into an exosome further loaded with folic acid to increase the tumor-targeting ability, and the indocyanine green-loaded exosome manufactured in this way overcomes the inherent shortcomings of indocyanine green, such as reduced stability in aqueous solutions, short blood circulation time, and lack of cancer targeting, and confirms that it can be effectively applied to SDT, thereby completing the present invention.

Maeda, H. et al., J. Controlled Release 2000, 65, 271-284. Antimisiaris, S. et al., Pharmaceutics, 2018, 10, 218.

The purpose of the present invention is to provide a novel nanocarrier for sonodynamic therapy and a method for producing the same, which is biocompatible and selectively delivers drugs to tumor sites while minimizing side effects on normal tissues and cells.

To achieve the above purpose, the present invention provides an exosome for tumor treatment loaded with an ultrasound sensitizer.

In the present invention, the ultrasonic sensitizer may be characterized by being at least one selected from the group consisting of porphyrin compounds, phthalocyanine compounds, rhodamine compounds, naphthalocyanines compounds, BODIPY compounds, and indocyanine green.

In the present invention, the exosome may be characterized in that a tumor-targeting substance is conjugated thereto.

In the present invention, the tumor-targeting agent may be characterized by being selected from the group consisting of folic acid, an antibody, a tumor-targeting peptide, an aptamer, cobalamin, vitamin A, vitamin C, or vitamin B1.

In the present invention, the exosome may be characterized by exhibiting anti-tumor activity through ultrasonic treatment.

The present invention also provides a pharmaceutical composition for treating tumors, comprising the exosome as an active ingredient.

In the present invention, the tumor may be characterized in that it is selected from the group consisting of osteogenic sarcoma, soft tissue sarcoma, breast cancer, lung cancer, brain cancer, bladder cancer, thyroid cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, stomach cancer, liver cancer, uterine cancer, kidney cancer, cervical cancer, skin cancer, esophageal cancer and ovarian cancer; Hodgkin's lymphoma, non-Hodgkin's, glioma, melanoma, myeloma, Wilms' tumor, acute lymphoblastic leukemia, acute myeloblastic leukemia, astrocytoma, glioma and retinoblastoma.

In the present invention, it may be characterized in that the composition is administered before ultrasonic irradiation.

In the present invention, the ultrasound may be characterized by being irradiated at a frequency of 0.1 to 15 MHz and an intensity of 0.01 to 1.0 W/cm ² for 30 to 300 seconds.

In the present invention, it may be characterized in that the ultrasound is irradiated 4 to 24 hours after administration of the composition.

In the present invention, the ultrasonic waves

(i) ROS production by ICG; and/or

(ii) It may be characterized by promoting ICG release from exosomes.

The present invention also provides a method for preparing exosomes loaded with an insoluble ultrasound sensitizer, comprising a step of incubating exosomes and an insoluble ultrasound sensitizer in PBS containing 1 to 5% (v/v) DMSO.

In the present invention, the manufacturing method may be characterized by mixing 1 x 10 ¹⁰ to 1 x 10 ¹² exosomes and 0.01 to 1.0 mg of an insoluble ultrasound sensitizer.

In the present invention, the exosome may be characterized by being conjugated with a tumor-targeting substance before or after mixing with an insoluble ultrasound sensitizer.

The exosomes loaded with ultrasound sensitizers according to the present invention are specifically accumulated in tumor cells and absorbed into cells, thereby minimizing side effects while effectively acting on tumors located deep within the body and inaccessible to photodynamic therapy, and do not cause in vivo toxicity due to the exosomes themselves, providing a non-invasive or minimally invasive treatment method for the treatment of various solid tumors including breast cancer.

Figure 1 is a schematic diagram of the preparation of FA-conjugated and indocyanine green-loaded exosomes (FA-ExoICG) and ultrasound-induced dynamic therapy. FA-ExoICG is selectively accumulated in tumors via the EPR effect and efficiently internalized into folate receptor-overexpressing breast cancer cells via folate receptor-mediated endocytosis. FA-ExoICG induces cell death by generating cytotoxic ROS in response to ultrasound.
Figure 2A shows the absorbance spectra of blank exosomes, free ICG, FA, FA-exosomes, and FA-ExoICG solutions. Figure 2B shows TEM images of blank exosomes and FA-ExoICG. The scale bar is 50 nm. Figure 2C shows Western blots of exosome markers CD63 and CD81 from various exosome samples. Figure 2D shows the changes in fluorescence intensity of free ICG, ExoICG, and FA-ExoICG after incubation in PBS at 4 °C for 10 days. Statistical significance was determined with respect to the ICG group. Figure 2E shows the ICG release profiles from ExoICG and FA-ExoICG in the absence or presence of ultrasound irradiation (1 min) at different incubation times. The ICG release from ExoICG and FA-ExoICG was promoted when exposed to ultrasound.
Figure 3A shows the relative cellular uptake of ICG in hDFB and MCF-7 cells treated with free ICG, ExoICG, and FA-ExoICG for 4 h. Figure 3B shows the quantification of ROS generation from free ICG, ExoICG, and FA-ExoICG in a cell-free system. Figure 3C shows the intracellular ROS levels in MCF-7 cells treated with free ICG, ExoICG, and FA-ExoICG before and after ultrasound irradiation. Figure 3D shows the cell viability of MCF-7 cells treated with free ICG, ExoICG, and FA-ExoICG before and after ultrasound irradiation (0.3 W/cm ² for 60 and 70 s). Figure 3E shows the cell viability of hDFB and MCF-7 cells treated with various samples under ultrasound irradiation. Figure 3F shows the cell viability of MCF-7 cells cultured with different concentrations of blank exosomes. Figure 3G shows the results of apoptosis rates in MCF-7 cells treated with free ICG, ExoICG, and FA-ExoICG.
Figure 4A shows the in vivo real-time biodistribution images at different time intervals after iv injection of free ICG, ExoICG, and FA-ExoICG into MCF-7 tumor xenograft mice. The fluorescence signal detected in the free ICG-injected mice was significantly lower than that in the other mice, such that the maximum value of the color scale bar in the fluorescence images of the ICG-treated mice (left images) was reduced by 12.5 times compared with that of the ExoICG- or FA-ExoICG-treated mice. Figure 4B shows the ex vivo images of each organ 24 h after administration of free ICG, ExoICG, and FA-ExoICG. Figure 4C shows the concentration-time profiles of ICG content in the plasma of tumor xenograft mice injected with single iv ExoICG and FA-ExoICG. Figure 4D shows the organ distribution of ICG in the whole organs of tumor xenograft mice after single iv injection of ExoICG and FA-ExoICG. Organ distribution of ICG 24 h after injection was expressed as a percentage of the injected dose (% ID/g). Data were expressed as mean ± SD (* p < 0.05, ** p < 0.01, n = 4).
Figure 5A shows the normalized tumor growth ratio as a function of time in MCF-7 tumor xenograft mice. Mice were exposed to ultrasound (0.5 W/cm ² ) for 3 min at 4 h after injection of each sample. Figure 5B shows representative images of tumors 14 days after intravenous injection of each sample and sonication for 3 min at 4 h after injection. Figure 5C shows the change in mouse body weight over time. Mice were sonicated for 3 min at 4 h after injection of each sample. Figure 5D shows H&E staining of mouse major organ slides 14 days after injection of various samples under ultrasound irradiation. The scale bar represents 200 μm.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

One of the major challenges in anticancer drug delivery is to selectively deliver therapeutic drugs to tumor sites while minimizing side effects on normal tissues and cells. To solve this problem, various types of synthetic nanocarriers, such as polymeric nanoparticles, liposomes, cell membrane-coated nanoparticles, and micelles, have been developed. These nanocarriers have been demonstrated to be able to transport therapeutic agents to tumor sites through the EPR effect. However, synthetic nanocarriers have had limitations in clinical application due to inherent toxicity and removal by the reticuloendothelial system (RES).

Unlike synthetic nanocarriers, exosomes, a type of cell-derived nanoparticle, have the advantages of high biocompatibility and long-term blood circulation. Therefore, in the present invention, exosomes were utilized as nanocarriers to efficiently and selectively deliver ultrasound sensitizers to tumors, and indocyanine green (ICG) as a therapeutic agent was loaded into exosomes as a nano ultrasound sensitizer. Encapsulating indocyanine green into exosomes could overcome the inherent shortcomings of indocyanine green, including aggregation and decomposition in aqueous solutions and lack of cancer specificity.

In the present invention, exosomes significantly improved the aqueous stability and cellular uptake of indocyanine green, thereby enhancing ROS generation under ultrasound irradiation. In particular, FA conjugation of exosomes further promoted the cellular internalization of indocyanine green into breast cancer cells. As a result, FA-ExoICG showed more sonotoxicity to MCF-7 breast cancer cells than to non-cancerous hDFB cells, demonstrating the sonodynamic effect targeted to cancer. Furthermore, the pharmacodynamic properties and tumor-targeted accumulation of indocyanine green were improved by loading it into exosomes. Exosomes loaded with indocyanine green could more intensively inhibit tumor growth in mice than free ICG. Specifically, mice treated with FA-ExoICG showed enhanced tumor localization of indocyanine green, thereby exhibiting a remarkable tumor growth inhibitory effect. In addition, FA-ExoICG was confirmed to have no side effects as it did not exhibit systemic toxicity, demonstrating that FA-conjugated and indocyanine green-loaded exosomes can be used as safe and effective nano-ultrasound sensitizers in cancer-targeting SDT and have significant potential for clinical applications.

Therefore, the present invention relates to exosomes for tumor treatment, in one aspect, loaded with an ultrasound sensitizer.

In the present invention, the ultrasonic sensitizer may be characterized by at least one selected from the group consisting of porphyrin compounds, phthalocyanine compounds, rhodamine compounds, naphthalocyanines compounds, BODIPY compounds, and indocyanine green, but is not limited thereto. The ultrasonic sensitizer is preferably indocyanine green.

In the present invention, the exosome may be characterized in that a tumor-targeting substance is conjugated thereto.

In the present invention, the tumor-targeting agent may be characterized by being selected from the group consisting of folic acid, an antibody, a tumor-targeting peptide, an aptamer, cobalamin, vitamin A, vitamin C, or vitamin B1, but is not limited thereto. The tumor-targeting agent is preferably folic acid.

In the present invention, the exosome may be characterized by exhibiting antitumor activity by ultrasonic treatment. In one embodiment, the exosome exhibits an antitumor effect by generating cytotoxic ROS by ultrasonic treatment.

From another aspect, the present invention relates to a pharmaceutical composition for treating tumors, comprising the exosome as an active ingredient.

In the present invention, the tumor includes both benign tumors and malignant tumors (cancer).

In the present invention, the tumor may be characterized by being selected from the group consisting of osteogenic sarcoma, soft tissue sarcoma, breast cancer, lung cancer, brain cancer, bladder cancer, thyroid cancer, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, stomach cancer, liver cancer, uterine cancer, kidney cancer, cervical cancer, skin cancer, esophageal cancer and ovarian cancer; Hodgkin's lymphoma, non-Hodgkin's, glioma, melanoma, myeloma, Wilms' tumor, acute lymphoblastic leukemia, acute myeloblastic leukemia, astrocytoma, glioma and retinoblastoma, but is not limited thereto.

In the present invention, it may be characterized in that the composition is administered before ultrasonic irradiation.

In the present invention, the ultrasound may be characterized by being irradiated at a frequency of 0.1 to 15 MHz and an intensity of 0.01 to 1.0 W/cm ² for 30 to 300 seconds, but is not limited thereto.

In the present invention, it may be characterized in that the ultrasound is irradiated about 4 to about 24 hours after administration of the composition, but is not limited thereto.

In the present invention, the ultrasonic waves

(i) ROS production by ICG; and/or

(ii) It may be characterized by promoting ICG release from exosomes.

In addition, the present invention successfully developed exosomes loaded with active cancer-targeting indocyanine green for safe and efficient ultrasound tumor treatment, and confirmed that exosomes with high indocyanine green loading efficiency can be produced using a simple culture method.

Therefore, the present invention, in another aspect, relates to a method for preparing exosomes loaded with an insoluble ultrasound sensitizer, comprising a step of incubating exosomes and an insoluble ultrasound sensitizer in PBS containing 1 to 5% (v/v) DMSO.

In the present invention, the manufacturing method may be characterized by, but is not limited to, mixing 1 x 10 ¹⁰ to 1 x 10 ¹² exosomes and 0.01 to 1.0 mg of an insoluble ultrasound sensitizer.

In the present invention, in order to improve cancer targeting, the exosome may be characterized by conjugating a tumor-targeting agent before or after mixing with the water-insoluble ultrasound sensitizer.

In the present invention, the water-insoluble ultrasonic sensitizer may be at least one water-insoluble ultrasonic sensitizer selected from the group consisting of porphyrin compounds, phthalocyanine compounds, rhodamine compounds, naphthalocyanines compounds, BODIPY compounds, and indocyanine green, and preferably indocyanine green.

In one embodiment, indocyanine green can be efficiently loaded into exosomes by mixing FA-exosomes and indocyanine green in PBS in the presence of DMSO, wherein DMSO increases the solubility of indocyanine green and its permeability through lipid membranes, thereby increasing the efficiency of indocyanine green loading into exosomes. In this case, when the concentration of DMSO is less than 1%, the loading efficiency is significantly low, and when it exceeds 5%, exosomes are substantially lost due to membrane destruction caused by DMSO, and thus the loading efficiency of indocyanine green is excellent in PBS containing 2 to 5% (v/v) DMSO. It is most preferable to use about 4% DMSO in PBS for indocyanine green encapsulation.

Recent studies have demonstrated that molecules involved in disease or cancer-related pathways are not increased in exosomes from HEK-293T cells, and that animal studies have generally demonstrated mild toxicity and immune responses, so exosomes are preferably isolated from human embryonic kidney HEK-293T cells, but are not limited thereto. However, it will be apparent to those skilled in the art that the use of other sources of exosomes, including milk and plants, may also be considered as alternative means for drug delivery.

The term "cultivation" in the present invention means a method of growing cells or microorganisms under appropriately artificially controlled environmental conditions.

In the present invention, it is preferable to replace the culture with a medium that does not contain fetal bovine serum, culture for 2 to 5 days, and then isolate exosomes.

In the present invention, the method for culturing the transformant can be performed using a method widely known in the art.

The above medium refers to a known medium used in animal cell culture, and can be selected from a group of commercially available serum free media, protein free media, and chemically defined media.

The above serum-free medium is a medium from which bovine serum components used in culturing animal cells have been removed, and SFM4CHO (HyClone) and EXCell (JHR Bioscience) are preferable. Insulin-like growth factor I (IGF-I), ethanolamine, ferric chloride, and phosphatidyl choline may be added, but are not limited thereto.

The above protein-free medium is an animal cell culture medium from which animal-derived proteins, particularly high-molecular-weight proteins having a molecular weight of 10 kDa or more, have been removed from a serum-free medium from which bovine serum components have been removed. ProCHO (Lonza) and PF-CHO (HyClone) may be selected, but are not limited thereto.

The above chemical definition medium is a medium for culturing animal cells without any animal-derived components and in which all components constituting the medium have known chemical structures, and may be selected from, but is not limited to, CDM4CHO (HyClone), PowerCHO2CD (Lonza), and CD-optiCHO (Life Technologies).

In another aspect, the exosomes described herein can also be used in methods of ultrasound dynamic therapy and, simultaneously, in vivo imaging (e.g., diagnostic imaging). In such methods, imaging can be used to monitor deposition and/or accumulation of exosome payload at a target site of interest.

For use in any of the methods described herein, the exosomes may generally be provided in a pharmaceutical composition together with at least one pharmaceutically acceptable excipient. Such compositions form a further aspect of the invention.

Pharmaceutical compositions for use in accordance with the present invention may be formulated using techniques well known in the art. The route of administration will depend on the intended use. Typically, they will be administered systemically and may therefore be provided in a form suitable for parenteral administration, for example, by intradermal, subcutaneous, intraperitoneal or intravenous injection. Suitable pharmaceutical forms include suspensions and solutions containing the exosomes together with one or more carriers or excipients. Suitable carriers include saline, sterile water, phosphate buffered saline and mixtures thereof.

The composition may additionally contain other agents such as emulsifiers, suspending agents, dispersing agents, solubilizers, stabilizers, buffers, preservatives, etc. The composition may be sterilized by conventional sterilization techniques.

Solutions containing exosomes can be stabilized, for example, by the addition of agents such as viscosity modifiers, emulsifiers, solubilizers, etc.

Preferably, the compositions for use in the present invention will be used in the form of an aqueous suspension in water or saline solution, for example, phosphate-buffered saline. The exosomes may be supplied in the form of a lyophilized powder for reconstitution at the point of use, for example, in water, saline or PBS.

The methods described herein comprise administering a therapeutically effective amount of a composition containing said exosomes. The exosomes are then distributed to a desired portion of the body or target body portion. Once administered to the body, the target body portion is exposed to ultrasound at a frequency and intensity that achieves the desired therapeutic effect. For exosomes loaded with indocyanine green, a typical activation procedure is schematically illustrated in the accompanying Figure 1. Treatment can be accomplished in a two-step process where the indocyanine green loaded exosomes are first ruptured by ultrasound, followed by the release of a sonosensitizer (SS) that can penetrate into the desired target tissue (e.g., a tumor). Subsequent ultrasound-activation of the sonosensitizer within the target cell results in the production of singlet oxygen, which can oxidize various cellular components, such as proteins, lipids, amino acids, and nucleotides, thereby destroying the target cell. Activation of the sonosensitizer typically occurs after delivery, i.e., upon release of the indocyanine green by the exosomes, but delivery of the exosomes and activation of the sonosensitizer can occur simultaneously.

The effective dosage of the compositions described herein will depend on the nature of the exosomes, the route of administration, the condition to be treated, the patient, etc., and can be adjusted accordingly.

The frequency and intensity of the ultrasound that can be used can be selected based on the need to induce activation of the indocyanine green loaded on the exosomes at the target site, the ultrasound frequency will typically be in the range of 0.1 to 15 MHz, preferably 0.1 to 3 MHz, which is used for medical ultrasound. The intensity (i.e. power density) of the ultrasound can be in the range of about 0.01 W/cm ² to about 10 W/cm ² , preferably about 0.1 to about 1 W/cm ² . The treatment time will typically be in the range of 10 seconds to 600 seconds, preferably 30 seconds to 300 seconds and this will depend on the intensity selected, i.e. for lower ultrasound intensities the treatment time will be longer and for higher ultrasound intensities the treatment time will be shorter. The ultrasound can be applied in a continuous or pulsed manner and can be delivered as a focused or columnar beam.

Any source capable of producing acoustic energy (e.g., ultrasound) may be used in the methods described herein. The source must be capable of directing the energy to a target site, and may include, for example, a probe or a device capable of directing the energy to the target tissue.

Example

Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Experimental method

1-1. Materials

Indocyanine green was purchased from TCI (Tokyo, Japan). FA, DCF-DA, dicyclohexylcarbodiimide (DCC), and NHS were purchased from Sigma-Aldrich (St. Louis, MO, USA). PD MiniTrap G-25 desalting columns were purchased from Cytiva (Marlborough, MA, USA). Slide-A-Lyzer Mini dialysis devices were supplied by Thermo Fisher Scientific (Waltham, MA, USA). Sonicator 740 (Mettler Electronics Corp., Anaheim, CA, USA) was used for sonication. Minimum essential medium/Earle's balanced salt solution (MEM/EBSS) and FBS were purchased from HyClone (Cytiva). Male CAnN.Cg-Foxn1 nu/Crl mice were provided by ORIENT (Gyeonggi-do, Korea).

1-2. Exosome isolation

Exosomes were produced from human embryonic kidney HEK-293T cells using ExoQuick-TC (System Biosciences, Palo Alto, USA). Briefly, cells were cultured for 3 days in the presence of exosome-free FBS for exosome production. Afterwards, the culture medium was cleared of cell debris by centrifugation (3000 g, 15 min). ExoQuick-TC solution was added to the supernatant and the mixture was kept overnight. The mixture was centrifuged at 1500 g for 30 min and the supernatant was removed. After a washing step, the pellet containing exosomes was reconstituted in PBS (pH 7.4). The final product was stored at -80 °C for further use. The concentration and size of the isolated exosomes were analyzed using NTA.

1-3. Western Blot Analysis

Exosomes were disrupted using cell lysis buffer, and protein amounts were measured using the BCA protein assay (Thermo Fisher Scientific). Proteins from the SDS-PAGE gel were transferred to polyvinylidene fluoride membranes. Exosomes were detected using antibodies to detect CD63 (MBL Life Science, Nagoya, Japan) and CD81 (Abcam, Cambridge, UK). Immunoreactive bands were observed using ECL Prime Western Blotting detection reagent (Cytiva). Images were acquired using the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

1-4. Preparation and characterization of ExoICG and FA ExoICG

Stock solutions of indocyanine green (ICG) and folic acid (FA) were prepared in DMSO (20 mg/mL for ICG and 100 mg/mL for FA). ExoICG was prepared by mixing 5 × 10 ¹¹ exosomes and 0.2 mg of indocyanine green in PBS containing 4% (v/v) DMSO and incubating for 2 h in the dark (4 °C). Unloaded indocyanine green was then removed using a PD MiniTrap G-25 desalting column.

FA-ExoICG was prepared by modifying the surface of exosomes with amine-reactive NHS ester-activated FA. NHS ester activation was synthesized by a previously reported method (Lee, R. J. et al. Biochim. Biophys. Acta, Biomembr. 1995, 1233, 134-144). Briefly, FA (0.20 g, 0.45 mmol) and DCC (0.18 g, 0.9 mmol) were mixed in DMSO (8 mL) in the presence of triethylamine (50.60 mg, 0.5 mmol). The mixture solution was kept under stirring in the dark at room temperature (RT) for 1 h. After adding NHS (0.11 g, 0.9 mmol), the mixture was stirred overnight at RT in the dark and then filtered to remove the insoluble byproduct dicyclohexyl urea. The resulting filtrate was precipitated using diethyl ether. Thereafter, the crude product was washed with tetrahydrofuran to obtain NHS ester-activated FA.

NHS ester-activated FAs were dissolved in 1% (v/v) DMSO containing PBS (1 mg/mL) and mixed with 2 × 10 ¹² exosomes at 4 °C. The mixture was stirred overnight to obtain FA-exosomes. The sample was then purified using a PD G-25 desalting column to extrude salts and free FAs. To obtain FA-ExoICG, FA-exosomes (5 × 10 ¹¹ exosomes) were mixed with 0.2 mg of indocyanine green in 4% (v/v) DMSO containing PBS and stirred for 2 h (4 °C) in the dark. The sample was purified using a PD G-25 desalting column and stored at 4 °C for further use. The conjugation efficiency of FAs to exosomes was quantified by UV-vis absorbance. FA-exosomes were dissolved in DMSO, and FA concentrations were quantified at 290 nm (Infinite M200 Pro, Tecan, Grodig, Austria). The molar number of FAs in exosomes was calculated using a standard curve of free FA solutions in DMSO. The number of FAs in single exosomes was determined based on the exosome counts of FA-ExoICG measured by NTA. The sizes of FA-exosomes and indocyanine green-loaded exosomes were determined by NTA at 25 °C. The morphologies of various exosome samples were observed by TEM (Talos F200X, FEI, Hillsboro, OR, USA). The absorbance spectra of free ICG, ExoICG, and FA-ExoICG were acquired using a spectrophotometer (Infinite M200 Pro). The efficiency of indocyanine green encapsulation into exosomes was measured using UV-vis absorbance and determined as follows: (amount of indocyanine green loaded) / (amount of indocyanine green initially added) × 100%.

1-5. Evaluation of colloidal stability of ExoICG and FA ExoICG

To assess the colloidal stability of FA-ExoICG in biological media, it was dispersed in PBS in the dark at 37 °C. Samples were obtained after 0, 1, 2, and 3 days of incubation for size measurement by dynamic light scattering analysis (Nano-ZS, Malvern, Worcestershire, UK).

1-6. Evaluation of aqueous stability of indocyanine green in ExoICG and FA-ExoICG

To investigate whether indocyanine green of ExoICG and FA-ExoICG is stable in aqueous solution over a long period of time, free ICG, ExoICG, and FA-ExoICG (20 μg/mL ICG) in PBS were maintained under normal lighting conditions at 4 °C for up to 10 days. After incubation for predetermined intervals, the fluorescence of indocyanine green was recorded (λex= 780 nm; λem= 835 nm).

1-7. In Vitro Drug release analysis

The release profiles of indocyanine green from ExoICG and FA-ExoICG were evaluated using a dialysis method. 100 mL of ExoICG (30 μg/mL ICG, 5.34 × ^10-10 exosomes/mL) and FA-ExoICG (30 μg/mL ICG, 5.65 × ^10-10 exosomes/mL) in PBS were loaded into a Slide-A-Lyzer Mini dialysis device (MW cutoff = 20,000 Da) and the device was maintained at 37 °C with shaking in the dark. The release profiles of indocyanine green from the samples were quantified in the same manner as previously reported. The amount of indocyanine green released from the samples was quantified based on the absorbance at 780 nm. To evaluate ultrasound-induced indocyanine green release from exosomes loaded with indocyanine green, ExoIC and FA-ExoICG were treated with ultrasound (1 MHz, 0.3 W/cm ² ) for 60 s and then kept at 37 °C in the dark. The amount of ICG released from ExoICG and FA-ExoICG after 2, 4, 8, and 24 h was quantified by UV-vis spectroscopy.

1-8. Cell culture

MCF-7 cells were cultured in MEM/EBSS containing 10% FBS and 1% antibiotics. hDFB cells were maintained in Dulbecco's modified Eagle's medium (DMEM, HyClone, Cytiva) supplemented with 10% FBS and 1% antibiotics.

1-9. Cellular uptake analysis of free ICG, ExoICG, and FA-ExoICG

MCF-7 and hDFB cells were cultured in 24-well plates (1 × 10 ⁵ cells/well). The cells were incubated with free ICG, ExoICG (40 μg/mL ICG, 7.13 × 10 ¹⁰ exosomes/mL), or FA-ExoICG (40 μg/mL ICG, 7.51 × 10 ¹⁰ exosomes/mL) and incubated for 4 h. For FA competition experiments, MCF-7 and hDFB cells were preincubated with free FA (5 mM) for 40 min prior to treatment with free ICG, ExoICG, and FA-ExoICG. Indocyanine green uptake into the cells was determined by the fluorescence intensity of the treated cells using a flow cytometer (CytoFLEX, Beckman Coulter, Brea, CA, USA). To investigate whether FA-ExoICG targets cancer cells more efficiently than hepatocytes, the cellular uptake of ExoICG and FA-ExoICG was quantified using MCF-7 and AML12 mouse hepatocytes. MCF-7 and AML12 cells were seeded in 24-well plates (1 × 10 ⁵ cells/well). After culturing the cells for 1 day, they were treated with ExoICG (40 μg/mL ICG, 7.13 × 10 ¹⁰ exosomes/mL) and FA-ExoICG (40 μg/mL ICG, 7.51 × 10 ¹⁰ exosomes/mL) for 4 h. After trypsinization of the cells, indocyanine green uptake by the cells was determined using a flow cytometer (CytoFLEX).

1-10. ROS generation by Free ICG, ExoICG, and FA-ExoICG during sonication

ROS production by indocyanine green-loaded exosomes in a cell-free system before and after sonication was quantified using DCF. DCF was obtained by adding KOH (0.1 mM) to DCF-DA. Briefly, suspensions of ExoICG (30 μg/mL ICG, 5.34 x ^10-10 exosomes/mL) and FA ExoICG (30 μg/mL ICG, 5.65 x ^10-10 exosomes/mL) in free ICG solution and DI water were treated with 10 μM DCF solution. The samples were then transferred to microcentrifuge tubes and subjected to 1 MHz ultrasound for 2 min at 0.3 W/cm ² . After an additional 1 h of incubation in the dark, ROS production was assessed based on the fluorescence signal of the liquid using a UV-vis spectrophotometer (Infinite M200 Pro, λex= 495 nm, λem= 529 nm).

After treatment with indocyanine green-containing exosomes, intracellular ROS levels were quantified using DCF-DA. Since DCF-DA is a ROS-detecting fluorescent dye, the green fluorescence intensity of DCF-DA-treated cells corresponds to the intracellular ROS levels of the cells. MCF-7 and hDFB cells were seeded in 12-well plates (3 × 10 ⁵ cells/well) and cultured for 1 day. The cells were then washed with PBS and incubated with DCF-DA solution (10 μM) for 30 min. The cells were then washed with PBS and treated with Free ICG, ExoICG (40 μg/mL ICG, 7.13 × 10 ¹⁰ exosomes/mL), or FA-ExoICG (40 μg/mL ICG, 7.51 × 10 ¹⁰ exosomes/mL) for 4 h. Afterwards, the cells were detached using 1 mL of MEM and divided into two groups. One group served as a control and the other group was exposed to 1 MHz (0.3 W/cm ² ) ultrasound for 1 min. After 30 min of incubation, the green fluorescence of the cells was quantified by flow cytometry.

1-11. In Vitro Sonotoxicity assessment

To investigate the sonication-induced cellular cytotoxicity of indocyanine green-loaded exosomes, the viability of MCF-7 cells treated with various samples in the presence or absence of ultrasound was measured using a standard MTT assay. MCF-7 and hDFB cells were seeded in 96-well plates (1 × 10 ⁴ cells/well). After the cells were cultured for 24 h, Free ICG (20 μg/mL), ExoICG (20 μg/mL ICG, 3.56 × 10 ¹⁰ exosomes/mL), and FA-ExoICG (20 μg/mL ICG, 3.76 × 10 ¹⁰ exosomes/mL) were added to the cells. After 4 h of treatment, the medium in the wells was replaced with culture medium. The cells were then exposed to 1 MHz ultrasound at 0.3 W/cm ² for 60 or 70 s and then cultured for 48 h. A conventional MTT assay was performed to determine the cell viability.

1-12. Apoptosis analysis using Annexin V-FITC/Propidium Iodide (PI) staining

MCF-7 and hDFB cells were seeded in 12-well plates (2 × 10 ⁵ cells/well). After the cells were cultured for 1 day, Free ICG, ExoICG (40 μg/mL ICG, 7.13 × 10 ¹⁰ exosomes/mL), or FA-ExoICG (40 μg/mL ICG, 7.51 × 10 ¹⁰ exosomes/mL) were added to each well and incubated for 4 h. The cells were then trypsinized and dispersed in PBS (0.5 mL). The cell suspension was transferred to a 0.6 mL microcentrifuge tube and sonicated at 0.3 W/cm ² for 1 min. After sonication, the cells were suspended in culture medium supplemented with 10% FBS. They were then immediately replated into 12-well plates and incubated for another 2 h. Cells were isolated according to the manufacturer's protocol (BD Biosciences, Heidelberg, Germany) and stained with Annexin V-FITC and PI. The percentage of apoptotic cells was quantified by flow cytometry (CytoFLEX). Annexin V-positive cells were determined as early and late apoptotic cells.

1-13. In Vivo Preparation of MCF-7 tumor xenograft mice for experiments

To establish tumor xenograft mice, MCF-7 cells (1 × 10 ⁶ cells) suspended in 100 μL of PBS were injected subcutaneously into the dorsal area of BALB/c nude mice (4 weeks old). When the tumor volume reached approximately 100 mm ³ , the mice were randomly divided into each treatment group with n = 4.

1-14. In vivo Biodistribution studies

Real-time biodistribution images of free ICG, ExoICG, and FA-ExoICG were acquired by intravenous injection of 100 μL of free ICG, ExoICG (1.0 mg ICG/kg, 1.78 × 10 ¹¹ exosomes/kg) or FA-ExoICG (1.0 mg ICG/kg, 1.87 × 10 ¹¹ exosomes/kg) into MCF-7 tumor xenografted mice (n = 4). Mice were euthanized 24 h after administration, and organs were harvested. Whole-body and major organ fluorescence images were acquired and analyzed by measuring indocyanine green fluorescence intensity (745-830 nm) at predetermined times within 24 h after injection using an Ami HT imaging system (Spectral Instruments Imaging, Tucson, AZ, USA). The acquired images were analyzed using Aura Imaging 2.20 software (Spectra Instruments Imaging).

1-15. In Vivo Pharmacokinetic studies

Free ICG (1.0 mg ICG/kg), ExoICG (1.0 mg ICG/kg, 1.78 × 10 ¹¹ exosomes/kg), and FA ExoICG (1.0 mg ICG/kg, 1.87 × 10 ¹¹ exosomes/kg) were administered by tail vein injection to MCF-7 tumor xenograft mice (n = 4). Blood samples (30 μL) were obtained from the saphenous vein at 2, 10, 30, 60, 120, 240, 480, and 1440 min after administration and stored in 0.5 mL heparinized polyethylene tubes. Plasma samples were collected by centrifugation (2000 g for 10 min at 4 °C) from the blood samples. Twenty-four hours after administration, tumors and major organs (brain, heart, liver, lung, spleen, and kidney) were collected, weighed, and homogenized. Plasma and tissue samples were kept at -80 °C for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. Plasma concentrations (ng/mL) were quantified using LC-MS/MS.

1-16. FA-ExoICG according to ultrasound radiation exposure In Vivo Anticancer activity

Tumor xenografted mice were injected iv via the tail vein and irradiated with ultrasound (n = 4): (i) PBS; (ii) PBS + ultrasound (0.5 W/cm ² ); (iii) free ICG (1.0 mg ICG/kg) + ultrasound; (iv) ExoICG (1.0 mg ICG/kg) + ultrasound; and (v) FA-ExoICG (1.0 mg ICG/kg) + ultrasound. The exosome concentrations of ExoICG and FA-ExoICG samples were 1.78 × 10 ¹¹ exosomes/mL and 1.87 × 10 ¹¹ exosomes/mL, respectively. Ultrasound exposure (1 MHz, 0.5 W/cm ² ) was performed at the tumor site for 3 min, 4 or 24 h post-injection. Tumor size was measured every other day and determined as follows: Tumor volume = (tumor length) × (tumor width) ² / 2. The body weight of each mouse was also measured every other day to evaluate the biological safety of the samples. After intravenous injection and ultrasound irradiation of each sample for 14 days, the tumor, heart, liver, lung, kidney, and spleen were collected. All tissues were stained with H&E to observe the histological results.

1-17. Statistical Analysis

All experiments were performed in triplicate unless otherwise specified. Data are expressed as mean ± standard deviation (SD). One-way ANOVA was used to evaluate statistical significance between experimental groups. Statistical significance was expressed as follows: *p <0.05; **p <0.01.

Example 2. Design, preparation, and characterization of indocyanine green-loaded exosomes

In the present invention, HEK-293T cell-derived exosomes with well-known molecular profiles were used, and FA was conjugated to the exosome surface to enhance the cancer specificity of HEK-293T cell-derived exosomes. To this end, exosomes (2.0 × 10 ¹² ) were mixed with 1 mg/mL N hydroxysuccinimide (NHS) ester-activated FA in 1% (v/v) dimethyl sulfoxide (DMSO)/phosphate buffered saline (PBS) and stirred overnight at 4 °C. Subsequently, FA-conjugated exosomes (FA-exosomes) were purified using a PD G-25 desalting column. Indocyanine green was efficiently loaded into exosomes by mixing FA-exosomes and indocyanine green in PBS in the presence of DMSO. DMSO was used to increase the solubility and permeability of indocyanine green through lipid membranes. A maximum concentration of 4% (v/v) DMSO in PBS was used for indocyanine green encapsulation because higher concentrations of DMSO resulted in substantial loss of exosomes due to membrane disruption by DMSO (Table 1).

To confirm the FA conjugation and indocyanine green encapsulation in exosomes, ultraviolet–visible (UV–vis) spectroscopy was performed. The UV–vis absorption spectra of blank exosomes, free FA, free ICG, FA-exosomes, and FA-ExoICG are shown in Fig. 2A. The absorption spectrum of FA-ExoICG showed characteristic peaks at ~290 and ~790 nm, which represent folic acid and indocyanine green, respectively, confirming that folic acid and indocyanine green were successfully incorporated into FA-exosomes. The conjugation ratio of FA to exosomes was determined to be 17.3% by quantifying the characteristic UV absorbance of FA at 290 nm. The density of FA to exosomes was 5.2 × 10 ⁵ FA/exosome. The encapsulation efficiencies of indocyanine green in ExoICG and FA-ExoICG were 64.9 and 60.7%, respectively. The loading capacities of indocyanine green for ExoICG and FA-ExoICG were 5.69 x 10 ^-10 and 5.32 x 10 ^-10 μg/exosome, respectively.

The hydrodynamic sizes and surface charges of blank and modified exosomes were determined by nanoparticle tracking analysis (NTA) (Nanosight NS300, Malvern, Worcestershire, UK) and zeta potential analysis (Nano-ZS Zetasizer, Malvern), respectively. The hydrodynamic sizes of blank exosomes, FA-exosomes, ExoICG, and FA-ExoICG were 108.5 ± 2.5, 115.7 ± 1.1, 136.0 ± 3.0, and 149.8 ± 3.4 nm, respectively (Table 2).

Exosomes with nano-sized diameters may have the additional advantage of enhanced cellular uptake and pharmacokinetics. The sizes of FA-exosomes were similar to those of blank exosomes, suggesting that FA modification did not induce exosome disruption or aggregation. The exosomes were slightly larger after loading with indocyanine green. Although most of the indocyanine green was encapsulated inside the exosomes, some of the indocyanine green may be embedded within the lipid bilayer of exosomes due to the lipophilic nature of indocyanine green, which may increase the exosome size. Zeta potential analysis revealed that all exosome-based samples had negative surface charges in the range of -21.4 to -28.5 mV (see Table 2 ). These minimal changes in the hydrodynamic diameter and surface charge of the surface-modified exosomes indicated that the intrinsic properties of the exosomes were maintained. The morphology of blank exosomes and FA-ExoICG was observed by transmission electron microscopy (TEM), and both blank exosomes and FA-ExoICG were confirmed to be spherical particles with a diameter of 40–60 nm (Fig. 2B).

The expression of exosome marker proteins CD81 and CD63 was analyzed by Western blotting to investigate the effects of surface modification and indocyanine green loading process on the protein content of exosomes. As shown in Fig. 2C, obvious bands representing CD81 and CD63 were observed in FA-exosomes, ExoICG, FA-ExoICG, and blank exosomes. These results demonstrate that the original protein content of exosomes is not significantly changed by surface modification and indocyanine green loading process.

Example 3. High colloidal stability of indocyanine green-loaded exosomes

To function successfully in vivo, drug carriers in biological fluids must have high colloidal stability. ExoICG and FA ExoICG suspended in PBS were incubated at 37 °C and their sizes were measured over various periods of time to investigate colloidal stability under saline physiological conditions.

As a result, ExoICG and FA-ExoICG showed no significant size change within up to 72 hours in PBS. This demonstrates the high colloidal stability of indocyanine green-loaded exosomes at high salt concentrations. It was found that indocyanine green-loaded exosomes can prolong the circulation time in the bloodstream due to their high colloidal stability and nano-size, and increase tumor accumulation through the EPR effect, thereby inducing efficient in vivo cancer-targeting SDT.

Example 4. Long-term aqueous stability of indocyanine green by encapsulation into exosomes

Indocyanine green decomposes in aqueous solution, resulting in simultaneous decrease in absorption and fluorescence. To investigate whether exosomes can provide long-term aqueous stability of indocyanine green, the fluorescence intensities of free ICG, ExoICG, and FA-ExoICG were monitored during culture in PBS at 4 °C for 10 days. Figure 2D shows that 83% of the initial fluorescence signal of ExoICG remained after 10 days of culture. FA-ExoICG showed almost the same fluorescence intensity change as ExoICG, indicating that FA modification of exosomes does not affect the long-term aqueous stability of exosomes. In contrast, the fluorescence signal of free ICG solution significantly decreased to 47% of the initial value after 10 days of culture (p < 0.01), confirming the low aqueous stability of indocyanine green. These results suggest that exosomes improved the stability of indocyanine green in aqueous solution.

Example 5. Ultrasound-responsive release of indocyanine green by indocyanine green-loaded exosomes

ExoICG and FA-ExoICG were irradiated with ultrasound (1 min, 0.3 W/cm ² ) prior to incubation in PBS (pH 7.4) at 37 ℃, and the ultrasound-responsive indocyanine green release was assessed. The amount of indocyanine green released from ExoICG and FA-ExoICG in the absence of ultrasound irradiation was not significantly different (Fig. 2E). In the absence of ultrasound irradiation, ExoICG and FA-ExoICG released 14.2 and 14.6% cumulative indocyanine green, respectively, within 24 h. When irradiated with ultrasound, significantly more indocyanine green was released from exosomes (p < 0.01). ExoICG and FA-ExoICG released 31.7% and 32.9% of indocyanine green, respectively, after 24 h of incubation with ultrasound.

These results suggest that ultrasound not only triggers ROS production in indocyanine green, but also acts as an external stimulus for the release of indocyanine green from target tumor tissues. In the present invention, it was confirmed that ultrasound irradiation induces destabilization of the exosome membrane, thereby releasing indocyanine green from exosomes, and thus exosomes can be used as effective ultrasound-responsive drug carriers.

To investigate whether increased ultrasound irradiation time promotes the release of indocyanine green from FA-ExoICG, the release rate of indocyanine green according to different ultrasound irradiation times was quantified. As a result, it was confirmed that more indocyanine green was released from FA-ExoICG as the ultrasound irradiation time increased. For example, when FA-ExoICG was irradiated with ultrasound for 70 s, 45.0% of indocyanine green was released within 24 h. It was found that more efficient SDT could be exhibited by increasing the released indocyanine green from exosomes.

Example 6. Cancer target cell uptake of FA-ExoICG

Free ICG, ExoICG, and FA-ExoICG with equivalent indocyanine green concentrations were incubated with folate receptor-overexpressing MCF-7 cells to investigate whether FA conjugation promotes cellular internalization of indocyanine green-loaded exosomes by breast cancer cells. Human dermal fibroblasts (hDFB) were employed as a folate receptor-negative control.

As a result, as shown in Fig. 3A, FA-ExoICG was more effectively taken up by folate receptor-overproducing MCF-7 cells than ExoICG without FA. In addition, the cellular uptake of FA-ExoICG by MCF-7 cells was found to be superior to that of FA-ExoICG by hDFB cells. These results confirmed the cancer-selective cellular internalization of FA-ExoICG.

To investigate the effect of FA on receptor-mediated endocytosis of FA-ExoICG, FA competitive binding studies were performed in which MCF-7 and hDFB cells were pre-incubated with free FA prior to ExoICG or FA-ExoICG treatment. When pre-incubated with free FA, the folate receptors of the cells were initially blocked by excess FA prior to FA-ExoICG treatment.

As shown in Fig. 3A, the preincubation with free FA decreased the cellular internalization of FA-ExoICG by MCF-7 cells. In contrast, the cellular internalization of FA-free ExoICG by MCF-7 cells was not significantly affected by the preincubation with FA. These results suggest that the enhanced uptake of FA-ExoICG by MCF-7 cells is causally related to folate receptor-mediated endocytosis.

Example 7. Enhanced phonodynamic effects by indocyanine green-loaded exosomes

The efficacy of SDT depends on the ability of ultrasound-sensitizing agents to generate ROS upon ultrasound exposure. Free ICG solution and ExoICG and FA-ExoICG suspensions were exposed to ultrasound (0.3 W/cm ² , 2 min) to investigate whether indocyanine green-loaded exosomes enhance the sonodynamic effects of indocyanine green. To investigate whether indocyanine green in ExoICG and FA-ExoICG generates ROS upon ultrasound exposure, the relative ROS production of free ICG, ExoICG, and FA-ExoICG in a cell-free system was measured before and after ultrasound irradiation (Fig. 3B).

Ultrasonic irradiation results showed that ROS production of free ICG, ExoICG, and FA-ExoICG was significantly increased. In particular, both ExoICG and FA-ExoICG showed stronger ROS production ability than free ICG. This result may be because the stability of indocyanine green in aqueous solution was improved due to exosome encapsulation (Fig. 2D). From the above results, it was found that exosomes loaded with indocyanine green were effective and stable nano ultrasound sensitizers for SDT compared to indocyanine green.

It is known that intracellular ROS production and oxidative stress by ultrasound sensitizers induce intracellular organelle dysfunction and cell death under ultrasound stimulation. After treatment of MCF-7 and hDFB cells with free ICG, ExoICG and FA-ExoICG under ultrasound stimulation, the intracellular levels of ROS were evaluated using the ROS-sensitive 2',7'-dichlorofluorescein diacetate (DCF-DA) dye. A stronger fluorescence signal in the dye-treated cells indicates a higher level of ROS production.

As a result, as shown in Fig. 3C, treatment with exosomes loaded with indocyanine green exhibited higher intracellular ROS levels compared to treatment with free ICG, regardless of sonication. This result may be due to the increased cellular internalization of indocyanine green by MCF-7 cells. In particular, cells treated with free ICG, ExoICG, or FA-ExoICG under ultrasound exposure showed a dramatic increase in intracellular ROS levels compared to cells that were not exposed to ultrasound (Fig. 3C). This result demonstrates that ultrasound triggers ROS production in exosomes loaded with indocyanine green. Cells treated with FA-ExoICG under ultrasound irradiation exhibited the highest intracellular ROS levels, which is interpreted as a result of the promoted cellular uptake due to FA conjugation. It was confirmed that MCF-7 cells cultured with FA-ExoICG exhibited higher intracellular ROS levels compared to hDFB cells, regardless of sonication. These results indicate that FA-ExoICG was better uptaken into MCF-7 cells than into hDFB cells, suggesting that FA-ExoICG may be an effective cancer-targeting nano-ultrasound sensitizer.

Example 8. Using FA-ExoICG In Vitro Cancer Targeted SDT

In vitro sonotoxicity of indocyanine green-loaded exosomes against folate receptor-negative hDFB and folate receptor-positive MCF-7 cells was investigated using a standard MTT assay. Cells were treated with 20 μg/mL free ICG, ExoICG (20 μg/mL ICG, 3.56 x ^10-10 exosomes/mL), and FA-ExoICG (20 μg/mL ICG, 3.76 x ^10-10 exosomes/mL) and then irradiated with ultrasound for 60 or 70 s. Cells were sonicated for up to 70 s because longer periods of ultrasound irradiation may induce cytotoxicity.

As a result, more than 88% of MCF-7 and hDFB cells maintained viability after 70 seconds of ultrasound irradiation, indicating that ultrasound irradiation caused minimal cytotoxicity in both MCF-7 and hDFB cells (Fig. 3D, 3E). Meanwhile, the viability of MCF-7 cells cultured with ExoICG was significantly decreased compared to that treated with free ICG (Fig. 3D). This is interpreted as an increase in cytotoxicity due to increased intracellular uptake of indocyanine green by exosomes. When cells were irradiated with ultrasound radiation and then treated with ExoICG or FA-ExoICG, cytotoxicity was significantly increased. Specifically, the viability of MCF-7 cells decreased from 66.9% after treatment with FA-ExoICG to 44.1% after 60 seconds of ultrasound irradiation (Fig. 3D), and the viability of cells treated with FA-ExoICG decreased more significantly after 70 seconds of ultrasound irradiation. In particular, the sonotoxicity of FA-ExoICG for MCF-7 cells was significantly greater than that of ExoICG, regardless of ultrasound irradiation. This result may be due to the cancer-specificity of FA-ExoICG. When the sonotoxicity of free ICG, ExoICG, and FA-ExoICG for MCF-7 and hDFB cells was compared, FA-ExoICG showed significantly more sonotoxicity for MCF-7 cells than for hDFB cells, whereas free ICG and ExoICG showed similar levels of sonotoxicity for both cell types. These results confirmed the cancer-specific sonotoxicity of FA-ExoICG.

Together with the evaluation of intracellular ROS production (Fig. 3C), the cytotoxicity evaluation confirmed that indocyanine green-loaded exosomes were effective nano-ultrasound sensitizers. The cytotoxicity of blank exosomes (i.e., exosomes without indocyanine green) against MCF-7 and hDFB cells was analyzed to evaluate the toxicity of the exosomes themselves. Blank exosomes did not show significant toxicity (>90% viability) up to a concentration of 2.0 × ^10-11 exosomes/mL against both MCF-7 and hDFB cells, confirming that exosomes are low-toxicity carriers (Fig. 3F). The exosome concentration used in the in vitro sonotoxicity study was approximately 3.8 × ^10-10 exosomes/mL to load an indocyanine green concentration of 20 μg/mL in ExoICG and FA-ExoICG, respectively, and blank exosomes at a concentration of 3.8 × ^10-10 exosomes/mL showed almost no cytotoxicity (Fig. 3F).

Therefore, we could confirm that the sonotoxicity of ExoICG and FA-ExoICG was not due to exosomes, but rather to indocyanine green contained in the exosomes. Considering that the inherent toxicity of drug carriers is a major obstacle to clinical use, the low toxicity of blank exosomes suggests that exosome-based nano-sonic sensitizers can be safely applied clinically to SDT.

Additionally, the degree of apoptosis in MCF-7 cells treated with free ICG, ExoICG, and FA-ExoICG was evaluated before and after ultrasound irradiation to investigate whether ultrasound induced sonotoxicity of ExoICG and FA-ExoICG. Under ultrasound irradiation, the early and late apoptotic populations were dramatically increased in cells treated with ExoICG or FA-ExoICG (34.09 and 57.65%, respectively) (Fig. 3G). Under ultrasound irradiation, FA-ExoICG treatment in particular induced a higher level of apoptosis than ExoICG (Fig. 3D).

The degree of cell death by FA-ExoICG treatment before and after ultrasound irradiation was also investigated for hDFB cells. When FA-ExoICG was treated after ultrasound irradiation, 31.80% cell death was induced, which was a significantly lower level than the cell death induced in MCF-7 cells under the same conditions. It is thought that the reason why FA-ExoICG treatment after ultrasound irradiation induces higher cell death in MCF-7 cells than in hDFB cells is because the intracellular ROS concentration is higher in MCF-7 cells than in hDFB cells.

Example 9. Effective intratumoral accumulation of FA-ExoICG

Nanoscale drug carriers, such as ~100 nm exosomes, can selectively accumulate in solid tumors because vascular permeability and lymphatic drainage are inhibited by the EPR effect. Hereinafter, we confirmed enhanced accumulation of indocyanine green delivered to the tumor area in the form of FA-ExoICG in MCF-7 tumor xenograft mice using in vivo NIR fluorescence imaging.

After intravenous administration of free ICG, ExoICG, and FA-ExoICG to mice, the biodistribution of free ICG, ExoICG, and FA-ExoICG in the mice body at 0-24 h was detected by the NIR fluorescence signal of indocyanine green using an in vivo imaging system (IVIS). All mouse groups detected fluorescence signals throughout the body 1 min after intravenous (i.v.) injection (Fig. 4A). Fluorescence of ExoICG and FA-ExoICG was detected in the tumor up to 24 h after injection, whereas the fluorescence of free ICG almost disappeared in the tumor within 24 h (Fig. 4A). These results confirmed that indocyanine green-loaded exosomes could effectively deliver indocyanine green to tumors via the EPR effect. In particular, the tumors of FA-ExoICG-administered mice showed higher fluorescence intensity than those of ExoICG-administered mice, confirming the excellent cancer specificity of FA-ExoICG. Meanwhile, ex vivo images of tumors and major organs collected 24 hours after injection also showed that indocyanine green accumulation in tumors in FA-ExoICG-treated and ExoICG-treated mice was significantly increased compared to mice treated with free ICG (Fig. 4B). These results suggest that indocyanine green-loaded exosomes are suitable for noninvasive NIR fluorescence imaging and can guide and monitor each treatment step simultaneously with in vivo SDT.

To investigate the intratumoral accumulation process of exosomes, the ratio of the fluorescence intensity of the tumor core area to the peritumoral tissue area (C/P) was calculated over time in free ICG-, ExoICG-, and FA-ExoICG-treated mice. The highest C/P ratio was observed at 4 h after injection in all groups, and it gradually decreased up to 8 h after injection. Therefore, in a study to achieve the most effective indocyanine green-mediated SDT in vivo, ultrasound was irradiated at 4 h after injection of each of the above samples.

The reason for the increase in the C/P ratio 24 hours after administration is that the amount of ICG remaining in the blood and peritumoral skin decreased more rapidly at 24 hours after administration than at 8 hours after administration [i.e., the ICG concentration in the peritumoral tissue area (P) decreased significantly].

Example 10. In vivo pharmacokinetics and biodistribution of indocyanine green-loaded exosomes

The in vivo pharmacokinetic properties and biodistribution of free ICG and indocyanine green-encapsulated exosomes were evaluated using an MCF-7 tumor xenograft mouse model. Temporal changes in blood indocyanine green concentrations were observed after intravenous administration of ExoICG and FA-ExoICG (1.0 mg ICG/kg).

The results are shown in Fig. 4C, and the pharmacokinetic parameters derived from the curves are summarized in Table 3. The blood indocyanine green concentrations of the ExoICG and FA-ExoICG groups showed similar patterns, but the initial indocyanine green concentration (C ₀ ) of the FA-ExoICG group was calculated to be 23.56 ± 5.21% of the injected dose (ID)/mL. This value was approximately 2-fold higher than that of the ExoICG group, indicating that FA-ExoICG was initially confined to the blood in contrast to the rapid accumulation of the ExoICG group in the tissues. In addition, the area under the curve (AUC) for 24 h in the FA-ExoICG group was 1.6-fold higher than that in the ExoICG group (Table 3). The elimination half-life [t _1/2 (z)] of FA-ExoICG was lower than that of the ExoICG group. Compared with ExoICG, tumor-targeted FA-ExoICG can approach and be absorbed by tumor tissues more rapidly than other tissues, which may result in a lower blood concentration of FA-ExoICG and a relatively shorter half-life in blood circulation than ExoICG. For the free ICG group, the C ₀ , elimination half-life, and AUC were 2.49 ± 0.73% ID/mL, 2.43 ± 0.59 h, and 0.69 ± 0.27% ID h /mL, respectively. In the free ICG group, the AUC for 24 h was approximately 5 times lower than that of the ExoICG group (Table 3), indicating that ExoICG maintained blood circulation longer than free ICG.

From the above results, it was found that exosomes can avoid elimination by the immune system and have prolonged blood circulation, so that exosomes loaded with indocyanine green can be clinically used as nano ultrasound sensitizers through long-term blood circulation.

After single intravenous administration of ExoICG and FA-ExoICG to tumor xenograft mice, the distribution of indocyanine green in the whole organs was investigated. The amount of indocyanine green in the tumor was measured to be 2.2% ID/g in ExoICG-treated mice and 8.2% ID/g in FA-ExoICG-treated mice at 24 h after injection, confirming that FA-ExoICG could target tumors more effectively than ExoICG (Fig. 4D). Similarly, the ex vivo biodistribution results obtained using IVIS also showed that the uptake of indocyanine green was higher in the lungs and liver of FA-ExoICG-treated mice than that of ExoICG-treated mice (Fig. 4B). FA-ExoICG showed the highest localization of indocyanine green in the liver at 24 h after administration (Fig. 4D).

Example 11. Effective in vivo sonodynamic cancer treatment effect by FA-ExoICG

To evaluate the in vivo synodynamic effect of indocyanine green-loaded exosomes using an MCF-7 tumor xenograft mouse model, each sample (PBS, free ICG, ExoICG, and FA ExoICG) was intravenously administered to the mouse model with solid tumors (~100 mm ³ ). The tumor area was irradiated with ultrasound (0.5 W/cm ² ) for 3 min at 4 or 24 h after administration, and the tumor volume change was measured for 14 days without additional injection or ultrasound irradiation.

As a result, 14 days after ultrasound irradiation, mice administered ExoICG or FA-ExoICG suppressed tumor growth more effectively than mice administered free ICG, and the FA-ExoICG treatment group was particularly superior in tumor growth inhibition effect compared to the other groups (Fig. 5A). The results of taking tumor images also showed that the FA-ExoICG treated mice had the smallest tumor size on day 14 (Fig. 5B), indicating that FA-ExoICG effectively accumulates in tumors, thereby exerting an excellent ultrasound dynamic therapeutic effect. Meanwhile, no change in body weight was observed in any treatment group for 14 days, indicating that the treatment had no side effects (Fig. 5C). In the tumors of the group administered with FA-ExoICG, nuclear shrinkage areas and fragmentation were clearly observed while examining with ultrasound (Fig. 5D). 14 days after administration of FA-ExoICG, the major organs of the mice were sectioned and stained with H&E, and each organ showed negligible pathological changes, confirming the high in vivo safety of FA-ExoICG (Fig. 5D).

It is well known that nanocarriers should be completely excreted from the body within a desirable time frame to prevent chronic side effects. It is known that macrophages in the liver and spleen mainly contribute to the clearance of foreign exosomes used as carriers, and ICG-loaded exosomes are expected to be eliminated from the body through the hepatic clearance pathway, as shown by the results of significant accumulation in the liver 24 h after administration. The ICG fluorescence intensity of the tumors of FA-ExoICG-treated mice was higher than that of ExoICG-treated mice regardless of the time after injection. In particular, the ratio of the amount of ICG in the tumors treated with FA-ExoICG to that of ExoICG-treated tumors was higher at 24 h after administration than at 4 h after administration. Therefore, the tumors were analyzed 24 h after injection to investigate whether the relatively increased amount of FA-ExoICG in the tumors compared to ExoICG could lead to the enhancement of SDT efficacy.

As a result, tumor growth was significantly inhibited in both ExoICG- and FA-ExoICG-treated mice compared to mice treated with free ICG. In particular, FA-ExoICG-treated mice showed significantly less increase in tumor volume over 14 days, which was similar to the tumor growth inhibition results using ultrasound irradiation at 4 hours after injection (Fig. 5A). In particular, compared to ultrasound irradiation 4 hours after administration, the relative tumor growth inhibitory effect of FA-ExoICG was significantly increased compared to that of ExoICG when ultrasound irradiation was performed 24 hours after administration (Fig. 5A).

Meanwhile, all mice did not show significant changes in body weight for 14 days, confirming the absence of therapeutic side effects. In addition, H&E staining images also confirmed that tumor tissues were destroyed more effectively in the FA-ExoICG treatment group than in the ExoICG treatment group. Meanwhile, pathological changes in the major organs of the ExoICG and FA-ExoICG-administered mice were almost negligible, confirming that ICG-loaded exosomes are safe nano ultrasound sensitizers.

While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A pharmaceutical composition for treating breast cancer, comprising an exosome loaded with indocyanine green and conjugated with folic acid as an active ingredient, wherein the composition is administered before ultrasound irradiation, and the ultrasound is irradiated once every 4 to 24 hours after administration of the composition.
delete delete delete A pharmaceutical composition according to claim 1, characterized in that the exosome exhibits antitumor activity upon sonication.
delete delete delete A pharmaceutical composition according to claim 1, characterized in that the ultrasound is irradiated at a frequency of 0.1 to 15 MHz and an intensity of 0.01 to 1.0 W/cm ² for 30 to 300 seconds.
delete In the first paragraph, the ultrasound
(i) ROS generation by indocyanine green; and/or
(ii) A pharmaceutical composition characterized by promoting the release of indocyanine green from exosomes.
A method for producing an exosome loaded with an insoluble ultrasound sensitizer, comprising a step of incubating the exosome and an insoluble ultrasound sensitizer in PBS (Phosphate-Buffered Saline) containing 1 to 5% (v/v) DMSO, wherein the exosome is characterized in that the exosome is loaded with indocyanine green as an insoluble ultrasound sensitizer and is an exosome to which folic acid is conjugated.
A method for producing exosomes, characterized in that in claim 12, 1 x 10 ¹⁰ to 1 x 10 ¹² exosomes and 0.01 to 1.0 mg of indocyanine green, a water-insoluble ultrasound sensitizer, are mixed.
A method for producing an exosome, characterized in that in claim 12, the exosome is conjugated with folic acid, a tumor-targeting substance, before or after mixing with an insoluble ultrasound sensitizer.