Method for preparing liposomes encapsulating anticancer agent including light irradiation, and pharmaceutical composition comprising same

Abstract

The present invention provides a method for preparing liposomes encapsulating an anticancer agent, the method comprising irradiation with blue light or near-infrared light. In addition, the present invention provides a pharmaceutical composition for treating cancer, comprising liposomes that encapsulate an anticancer agent and are obtained by the preparation method.

Description

Method for preparing liposomes encapsulating anticancer agents including photoirradiation and pharmaceutical compositions comprising the same The present invention relates to a method for preparing liposomes encapsulating anticancer agents, including photoirradiation, and a pharmaceutical composition comprising the same. More specifically, the present invention relates to a method for preparing liposomes encapsulating anticancer agents, including irradiation with blue light or near-infrared light; and a pharmaceutical composition for treating cancer, comprising liposomes encapsulating anticancer agents obtained by the method. Cell-based therapies targeting specific cancer cells have shown great promise in cancer treatment. However, the efficacy of these drugs can be limited by cancer patients' compensatory or drug-elimination mechanisms. Therefore, high doses of the drug may be required to overcome these limitations, which can lead to drug resistance in cancer cells or adverse effects in normal cells, limiting the efficacy of many cancer treatments. Combination therapy (also known as cocktail therapy), which involves administering two or more drugs at low doses, overcomes some of these limitations and enhances the efficacy of cancer treatments. Doxorubicin (DOX) and paclitaxel (PTX) are commonly used in the treatment of metastatic breast cancer. However, their combination has shown suboptimal clinical outcomes due to an increased risk of cardiotoxicity. Therefore, there is a need to reduce the toxicity of existing effective drug combinations. Co-formulated drugs can be influenced by the drug delivery system used. Therefore, drug delivery systems should be optimized to maximize target cell cytotoxicity while minimizing the potential for drug resistance and off-target effects. To achieve this, the use of various drug delivery systems has been evaluated while observing synergistic effects between the administered drugs [Y. Li, H. et al., Drug Deliv 2020, 27, 1044; W. Yin, et al., J Drug Deliv Sci Technol 2022, 69; Y. Liu, et al., Mol Pharm2014, 11, 1651-1661; S. Lou, et al., ACS Omega2018, 3, 9210; J. Yu, et al., Acta Pharm Sin B2020, 10, 1730; MS Franco, et al., Biomed Pharmacother2019, 109, 1728]. In addition, in a previous study, PEG-polypeptide nanovehicles were used to deliver DOX and PTX to non-small cell lung cancer patients [S. Lv, Z. Tang, et al., Biomaterials2014, 35, 6118]. In addition, redox-sensitive micelles were used to co-deliver PTX and DOX to skin cancer lesions [H. Nsairat, et al., Heliyon2022, 8, e09394]. Among the drug delivery systems used, liposomes have shown an excellent ability to encapsulate multiple drugs with varying degrees of hydrophobicity and deliver them to the same site of action [H. Nsairat, et al.,Heliyon2022,8, e09394; M. B. R. Pierre, et al.,Arch Dermatol Res2011,303, 607]. However, existing liposomal formulations are not optimized for loading excessively hydrophobic drugs, which limits their loading efficiency and their use for anticancer drug delivery [M. Kolter, et al., Eur J Pharm Biopharm 2019, 140, 20; MD Fulton, et al., Int. J. Mol. Sci. 2023, 24, 6615; S. Naderinezhad, et al., RSC Adv. 2017, 7, 30008]. Therefore, there is a need to develop stable liposomal formulations with improved drug loading capacity. Extensive research has been conducted to produce liposomes that meet these requirements by manipulating natural and synthetic phospholipids using factors such as pH, halogen reactions, and magnetic fields [D. Pasarin, et al., Polymers (Basel) 2023, 15, 782; [P. Liu, G. et al.,Molecules2022,27,1372; OV Zaborova, et al.,J Colloid Interface Sci2023,640,558; H. Son, et al.,RSC Adv.2023,13,9788; PS Zangabad, et al.,Nanotechnol Rev2017,7,95; Y. Lee, et al.,Wiley Interdiscip Rev Nanomed Nanobiotechnol2017,9,1450]. For example, magnetic anionic liposomes have been designed for efficient targeting [Y. Kono, et al.,Drug Deliv2017,24,1740]. Furthermore, liposomes that are sensitive to reactive oxygen species levels and can respond to abnormal tissue environments have been developed for effective cancer therapy. However, the use of physical stimuli such as electromagnetic waves for drug delivery can disrupt the structure of the lipid layer and reduce the function of liposomes as drug delivery vehicles [A. Mickova, et al.,ACTA VET. BRNO2008,77, 285]. The present inventors conducted various studies to develop a method for improving the encapsulation level of anticancer agents within liposomes (i.e., the encapsulation efficiency of the anticancer agent) and enhancing the delivery capacity of anticancer agent-loaded liposomes. As a result, the present inventors discovered that anticancer agent-encapsulated liposomes (i.e., anticancer agent-loaded liposomes) obtained by irradiating liposomes with blue or near-infrared light and then encapsulating the anticancer agent not only exhibit high anticancer agent encapsulation efficiency but also increase the retention time of the anticancer agent in cancer cells, thereby sustaining high antitumor activity. Accordingly, the present invention aims to provide a method for preparing liposomes encapsulating anticancer agents, which comprises irradiating them with blue light or near-infrared light. In addition, the present invention aims to provide a pharmaceutical composition for treating cancer, which comprises, as an active ingredient, a liposome in which an anticancer agent obtained by the above manufacturing method is encapsulated. In one aspect of the present invention, a method for preparing a liposome encapsulating an anticancer agent is provided, comprising: (a) irradiating a liposome with blue light or near-infrared light; and (b) encapsulating an anticancer agent in the light-irradiated liposome obtained in step (a). In the manufacturing method of the present invention, the liposome can be formed from a mixture of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]. The molar ratio of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] in the mixture can be 10 to 25:1. In the manufacturing method of the present invention, the blue light may have a wavelength of 400 to 495 nm, and the near-infrared light may have a wavelength of 780 to 2,500 nm. In addition, the light irradiation may be performed for 0.5 to 2 hours. The above anticancer agent may be doxorubicin or a pharmaceutically acceptable salt thereof; paclitaxel or a pharmaceutically acceptable salt thereof; or a combination of doxorubicin or a pharmaceutically acceptable salt thereof and paclitaxel or a pharmaceutically acceptable salt thereof. In another aspect of the present invention, a pharmaceutical composition for treating cancer is provided, which comprises, as an active ingredient, a liposome encapsulating an anticancer agent obtained by the above manufacturing method. In the pharmaceutical composition of the present invention, the cancer may be breast cancer, non-small cell lung cancer, or skin cancer. According to the present invention, it has been revealed that liposomes encapsulating anticancer agents obtained by irradiating liposomes with blue light or near-infrared light and then encapsulating anticancer agents (i.e., anticancer agent-loaded liposomes) not only exhibit high anticancer agent encapsulation efficiency, but also increase the retention time of the anticancer agent in cancer cells, thereby sustaining high antitumor activity. Therefore, liposomes encapsulating anticancer agents obtained according to the manufacturing method of the present invention can be usefully applied to the treatment of cancers such as breast cancer, non-small cell lung cancer, and skin cancer. Figure 1 shows the characterization results of liposomes loaded with DOX and PTX using conventional and LED irradiation methods. A: Schematic diagram of three methods used to load drugs into liposomes. DOX and PTX were loaded into NsLipo using conventional methods, while drugs were loaded into LsLipo@B and LsLipo@N using blue and near-infrared (NIR) LED wavelengths, respectively. B: Dynamic light scattering (DLS) measurements of the sizes of NsLipo, LsLipo@B, and LsLipo@N. C: Zeta potential measurement results of NsLipo, LsLipo@B, and LsLipo@N. D: Scanning electron microscopy (SEM, left) and transmission electron microscopy (TEM, right) images of NsLipo, LsLipo@B, and LsLipo@N. E: Fluorescence images of DOX (red) and PTX (green) loaded into NsLipo, LsLipo@B, and LsLipo@N. F: Quantitative analysis results of drug loading efficiency of NsLipo, LsLipo@B and LsLipo@N. G: Drug release profiles of DOX and PTX from NsLipo, LsLipo@B, and LsLipo@N over time. Figure 2 shows the results of measuring changes in the chemical structure of lipids after LED stimulation. A: Before and after LED irradiation 1 H-NMR spectrum. B: FTIR spectra of lipids before and after LED irradiation. C: Results of a comparison of the distance between lipids using BIOVIA Materials Studio software simulations. Because deprotonation occurs as a result of LED irradiation, the nitrogen atoms, and therefore the distance between lipids, increase. Figure 3 shows the effect of LED irradiation on the interaction between duramycin and liposomes. A: Results of confirming chemical structural changes induced by LED irradiation in liposome membranes. Using Cy5-conjugated duramycin, which specifically binds to ethanolamine, we observed the interaction between duramycin and ethanolamine in liposomes. This analysis demonstrated that LED irradiation alters the chemical structure of the liposome membrane, preventing duramycin from binding to ethanolamine. B: Quantitative assessment of the interaction between duramycin and NsLipo, LsLipo@B or LsLipo@N. C: Confocal microscopy images of Cy5-duramycin bound to NsLipo and LsLipo. The red box indicates the area where fluorescence intensity was measured. D: Visualization of membrane structural changes induced by LED irradiation using DiI and DiO. NsLipo and LsLipo were labeled with hydrophobic fluorescent dyes DiI (red) and DiO (green). Figure 4 shows the results of evaluating the effect of LED irradiation on the structure and mechanical properties of liposomes. A: AFM image showing structural changes induced by LED irradiation in liposomes. B: Results of Young's modulus measurement of liposomes before and after LED irradiation. C: AFM images of drug-loaded liposomes before and after LED irradiation. Figure 5 shows the results of evaluating the cancer cell apoptosis-inducing ability of LsLipo and NsLipo. A: Fluorescence microscopy images demonstrating the drug delivery efficacy of LsLipo and NsLipo in cancer cells. The fluorescence intensity of cancer cells treated with DOX- and PTX-loaded liposomes generated using LED irradiation or conventional methods was measured using flow cytometry at different time points. B: Flow cytometry analysis was performed to confirm the intracellular delivery of DOX and PTX by LsLipo and NsLipo. C: After treatment of MCF-7 cells with LsLipo and NsLipo loaded with DOX and PTX, cancer cell death was observed in vitro. D: Fluorescence microscopy images of PTX and DOX in cancer cells 24 and 48 hours after liposome treatment (left) and quantitative analysis of intracellular PTX- and DOX-derived fluorescence (right). E: Quantitative analysis of the fluorescence intensity of intracellular PTX and DOX at 24 and 48 hours. F: Graph showing cancer cell death over time in each treatment group. Figure 6 shows the results of evaluating the effects of LsLipo and NsLipo loaded with anticancer drugs on tumors in model mice. A: Schematic diagram showing the dosing schedule for tumor-bearing mice loaded with NsLipo@B and LsLipo@N drugs. B: Representative images of whole tumors from nude mice treated with DPBS (negative control), NsLipo, LsLipo@B, or LsLipo@N. C: Tumor growth curves of the control and liposome-treated groups. Tumor volume (mm 3 ) is (tumor length [mm] X tumor width [mm 2 ]) / 2 formula. Each point represents the mean ± standard deviation of five animals. D: Comparison of tumor weights in the four treatment groups. E: Comparison of tumor morphology in the four treatment groups. F: Comparison of mouse body weights during the 28-day treatment period. G: H&E (upper panel) and TUNEL (lower panel) staining images of tumor sections. Green arrows in the H&E image and white arrows in the TUNEL image indicate the locations of detached and necrotic tumor tissue. Figure 7 shows the encapsulation of DOX and PTX at various concentrations. The amount of each drug encapsulated in the liposomes increased dose-dependently (n=3). The graph shows how the fluorescence intensity changes with the concentration of DOX (upper panel) and PTX (lower panel). NS, not significant; *p<0.05; **p<0.01; ***p<0.001, and ****p<0.0001. Figure 8 shows the encapsulation of drugs other than DOX and PTX into liposomes. The liposomal encapsulation of substances with molecular weights (MWs) different from DOX and PTX was evaluated. The left graph shows the loading efficiency of FITC-dextran (average MW: 4,000 g / mol), which has a high molecular weight, and the right graph shows the loading efficiency of TRITC (MW: 444 g / mol), which has a low molecular weight (n=3). ****p<0.0001 Fig. 9 is 1Shows the integral values ​​of liposomal lipids determined using H-NMR. 1 The integrals of PE(a) and DSPE-PEG-2k(b) derived using H-NMR are plotted in the graph. The x-axis of the graph represents chemical shifts in ppm. The H at 5.2 ppm, indicated by the red arrow, was used as a reference (integration value: 1) for comparing H values ​​at other lipid sites. The peaks at 3.7 and 3.2 ppm represent the lipid head region, and the peak at 1.2 ppm represents the lipid tail region. Figure 10 shows a CLSM image of liposomes interacting with duramycin. Figure 11 shows the zeta potential of duramycin-treated liposomes. Fig. 12 is 1 The partitions of the H-NMR spectra are shown. The regions highlighted in yellow are obtained from the head regions of PE (a) and DSPE-PEG-2k (b). 1 H-NMR results are shown. Red numbers represent the integrals of H at the corresponding positions. Figure 13 shows CLSM images of MCF-7 cells taken at different time points after liposome treatment. Red fluorescence represents DOX and green fluorescence represents PTX within MCF-7 cells. Figure 14 shows the uptake of DOX- and PTX-loaded liposomes by MCF-7 cells. Liposome uptake by MCF-7 cells was measured by flow cytometry; DOX fluorescence (left) and PTX fluorescence (right) were measured at 2-hour intervals starting 4 hours after treatment. Figure 15 shows the results of apoptosis analysis of MCF-7 cells. Apoptosis of MCF-7 cells was analyzed by flow cytometry at different time points using Annexin V and PI staining. The lower right panel shows early apoptosis, and the upper right panel shows late apoptosis. Measurements were performed at 2-hour intervals, starting 4 hours after treatment. Liposomes are widely used as drug delivery nanoplatforms due to their versatility and biocompatibility. However, their ability to load specific drugs, such as anticancer agents, remains unsatisfactory. We prepared liposomes using a combination of 18:0 polyethylene glycol (PE) and DSPE-PEG-2k lipids and loaded them with doxorubicin (DOX) and paclitaxel (PTX). To investigate the effect of light-emitting diodes (LEDs) on liposome structure and drug loading efficiency, we investigated the effects of LEDs on liposome structure and drug loading efficiency. Scanning and transmission electron microscopy revealed that the surfaces of liposomes irradiated with blue or near-infrared LEDs (LsLipo) were rougher and more irregular than those of liposomes not irradiated with LEDs (NsLipo). Nuclear magnetic resonance (NMR) analysis revealed that the hydrogen peaks derived from lipid head groups were lower in LsLipo than in NsLipo, indicating that LED irradiation altered the chemical and physical properties of the liposomes. Structural changes, such as a decrease in stiffness induced by LED irradiation, increased the loading efficiency of DOX and PTX. In vitro and in vivo experiments showed that LsLipo was more effective than NsLipo in inhibiting cancer cell growth. These results suggest that LED irradiation enhances the drug delivery efficacy of liposomes and offers new possibilities for improving drug delivery systems. The present invention provides a method for preparing liposomes encapsulating anticancer agents, comprising the steps of (a) irradiating liposomes with blue light or near-infrared light; and (b) encapsulating anticancer agents in the light-irradiated liposomes obtained in step (a). Liposomes have a lipid bilayer structure formed from, for example, cationic, anionic, and / or neutral lipids. The liposomes can be formed from a variety of lipids, including, but not limited to, one or more lipids selected from the group consisting of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]. In one embodiment, the liposomes can be formed from a mixture of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]. In the above mixture, the molar ratio of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] may be 10 to 25:1, preferably 12 to 20:1, and more preferably about 15:1. The formation of the liposomes can be accomplished by various methods commonly used in the art, such as the hydration method. For example, liposomes can be formed by dissolving a lipid or lipid mixture in an organic solvent (e.g., chloroform), vaporizing the organic solvent to form a film-like lipid, hydrating the suspension with an aqueous medium such as phosphate-buffered saline, and then sonicating the resulting suspension. The irradiation with the blue light or near-infrared light can be performed by irradiating the liposome (e.g., a liposome suspension in an aqueous medium) using a light emitting diode (LED) having an appropriate wavelength. The blue light can have a wavelength of 400 to 495 nm, preferably a wavelength of 430 to 470 nm, and more preferably a wavelength of about 450 nm. In addition, the near-infrared light can have a wavelength of 780 to 2,500 nm, preferably a wavelength of 780 to 1,000 nm, and more preferably a wavelength of about 800 nm. The light irradiation can be performed for 0.5 to 2 hours, and preferably for about 1 hour. The present invention includes a step [i.e., step (b)] of encapsulating an anticancer agent in the photo-irradiated liposomes obtained in step (a). The encapsulation can be performed by any encapsulation method commonly used in the art. For example, encapsulation can be performed by (i) adding a solution obtained by dissolving an anticancer agent in a water-miscible organic solvent to a solution containing the photo-irradiated liposomes obtained in step (a) in an aqueous solvent, and then (ii) stirring the resulting solution for 2 to 2.5 hours. The aqueous solvent includes, for example, phosphate-buffered saline. The water-miscible organic solvent may be any solvent capable of dissolving an anticancer agent, and includes, without limitation, dimethyl sulfoxide (DMSO), ethanol, and the like. The stirring can be performed for 2 to 2.5 hours at room temperature or under heated conditions, but is not limited thereto. If necessary, the anticancer agent not encapsulated in the liposomes can be removed by a conventional method such as centrifugation. In one embodiment, the anticancer agent may be doxorubicin or a pharmaceutically acceptable salt thereof; paclitaxel or a pharmaceutically acceptable salt thereof; or a combination of doxorubicin or a pharmaceutically acceptable salt thereof and paclitaxel or a pharmaceutically acceptable salt thereof. Preferably, the anticancer agent may be a combination of doxorubicin or a pharmaceutically acceptable salt thereof and paclitaxel or a pharmaceutically acceptable salt thereof. The present invention also provides a pharmaceutical composition for treating cancer, which comprises, as an active ingredient, a liposome encapsulating an anticancer agent obtained by the above manufacturing method. Cancers to which the pharmaceutical composition of the present invention can be applied include various cancers depending on the encapsulated anticancer agent. In one embodiment, the anticancer agent may be a combination of doxorubicin or a pharmaceutically acceptable salt thereof and paclitaxel or a pharmaceutically acceptable salt thereof, and the cancer may be breast cancer, non-small cell lung cancer, or skin cancer. The pharmaceutical composition of the present invention can be formulated into various dosage forms that include, in addition to liposomes encapsulating the anticancer agent obtained by the above manufacturing method as an active ingredient, a pharmaceutically acceptable carrier. That is, the pharmaceutical composition of the present invention can be in the form of a preparation for parenteral administration such as an injection, for example, a form suitable for intramuscular, intraperitoneal, subcutaneous, or intravenous administration, and can be in a single-dose or multiple-dose dosage form. The injection is usually prepared as a sterile solution of liposomes encapsulating the anticancer agent obtained by the above manufacturing method of the present invention, and the pH of the solution should be suitably adjusted and buffered. In the case of intravenous administration, the total concentration of the solute should be adjusted to impart isotonicity to the formulation. The pharmaceutical composition can be in the form of an aqueous solution containing a pharmaceutically acceptable carrier, such as saline having a pH of 7.4. In one embodiment, the pharmaceutical composition of the present invention can be administered once or multiple times at a known dosage for the anticancer agent encapsulated in the liposome. For example, when the anticancer agent encapsulated in the liposome is a combination of doxorubicin or a pharmaceutically acceptable salt thereof and paclitaxel or a pharmaceutically acceptable salt thereof, the liposome encapsulating the anticancer agent may be administered once every three days or multiple times at a dosage of 50 to 150 mg / kg, for example, 90 to 100 mg / kg. Of course, the dosage and frequency of administration may be appropriately adjusted depending on the patient's condition, age, severity, etc. Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention and the present invention is not limited to them. Example 1. Materials and experimental methods (1) Material 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](DSPE-PEG

[2000] -amine) and 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine (PE) were purchased from Avanti Polar Lipid (Alabaster, AL, USA) and used without further purification. Oregon Green®-488-conjugated paclitaxel (PTX) (Flutax-2; Cat no. P22310) and eBioscience TM The Annexin V Apoptosis Detection Kit (Cat. no. 88-8007-74) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Doxorubicin hydrochloride (DOX; Cat. no. D1515) and semisynthetic PTX (Cat. no. T7191) were purchased from Sigma Aldrich (St. Louis, MO, USA). Cy5-duramycin was purchased from Molecular Targeting Technologies, Inc. (West Chester, PA, USA) and used directly without further purification. The MCF-7 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle's medium, low glucose (DMEM-low), fetal bovine serum (FBS), and Dulbecco's phosphate-buffered saline (DPBS) were purchased from HyClone (Logan, Canada). Antibiotic-antimycotic solution and trypsin-EDTA were purchased from Gibco (Waltham, MA, USA). (2) Preparation of LED-irradiated and non-irradiated liposomes Liposomes were prepared using a hydration method. Briefly, lipid powder (PE:DSPE-PEG-2k, molar ratio 15:1) was first heated to above 100°C in chloroform. After evaporation of the chloroform, the resulting film-like lipid was hydrated with DPBS to obtain a 10 mg / mL suspension. The suspension was sonicated for 3 min in a waterbath sonicator, and the liposomes were then irradiated with blue and NIR LEDs for 1 h. After LED stimulation, dual drug loading was performed with 580 ng DOX and 132 ng PTX (conjugated to Oregon Green) dissolved in 1 μl of DMSO per 100 μg of lipid powder. The mixture was encapsulated at room temperature for 2 h. Free drug was removed by centrifugation (12,150 × g, 4°C, 30 min). (3) Characteristic analysis of NsLipo and LsLipo The size and surface charge were measured by dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Panalytical, Malvern, UK). The morphologies of NsLipo, LsLipo@B, and LsLipo@N were observed using SEM and TEM. SEM was performed at APRO Inc. (Seoul, Korea), and TEM was performed at Eulji University (Seongnam, Korea). (4) Fluorescence detection Fluorescence of liposomes was analyzed using a microplate reader (SpectraMax iD5, Molecular Devices) and CLSM (Olympus FV3000, Tokyo, Japan). Liposome solutions containing drugs were loaded into 96-well plates, detected at 480 / 590 nm for DOX and 490 / 530 nm for PTX, and quantitatively analyzed using SoftMax Pro software. Liposome morphology was determined from fluorescence images captured using CLSM. DOX was detected using a tetramethylrhodamine-isothiocyanate (TRITC) laser, and PTX was detected using an Oregon Green 488 laser. (5) Lipid analysis Changes in lipid chemical structure can be detected by proton nuclear magnetic resonance ( 1 H-NMR; 600 MHz FT-NMR, Delta V6.1.0 software, JEOL Ltd.) and FTIR (IRspirit, Shimadzu, Japan) were used for analysis. Each lipid preparation was dissolved in chloroform-d and irradiated with LED in a liquid state. 1 H-NMR spectra were recorded and analyzed using Delta V 6.1.0 software. FTIR analysis was performed to detect changes in the chemical bonding of the amine group. (6) Atomic force microscopy analysis Drug-free and drug-loaded liposome samples were analyzed using an atomic force microscope (AFM) (JPK NanoWizard ULTRA Speed, Bruker) in a dry state on silicon wafers. Data were analyzed using XEI software (Park Systems). The surface state and height of the liposomes were determined using AFM. Young's modulus was also determined for the drug-loaded liposomes. AFM was performed at the Korea Advanced Nano Fab Center (Suwon, South Korea). (7) Cell culture The human breast cancer cell line MCF-7 was cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS) (HyClone) and 1% antibiotic-antimycotic (Gibco). The human breast cancer cell line MDA-MB-231 was cultured in DMEM (HyClone) supplemented with 10% FBS (HyClone) and 1% antibiotic-antimycotic solution (Gibco). All cells were cultured at 37°C and 5% CO2. (8) Cell uptake and apoptosis MCF-7 cells were treated with NsLipo, LsLipo@B, or LsLipo@N; then, liposome uptake by the cells was monitored at 2-h intervals, starting 4 h after treatment. Single cell suspensions (4 Х 10 5 cells / ml) were prepared and immediately analyzed by flow cytometry (Guava Technologies). Annexin V- and propidium iodide (PI)-stained MCF-7 cells were quantified by flow cytometry to assess the level of apoptosis. Briefly, MCF-7 cells were dissociated and prepared as single-cell suspensions in T / E buffer. Single-cell suspensions (5 Х 10 5(cells / ml) were stained with annexin V APC for 15 min at room temperature in the dark, followed by PI staining. Cells were then acquired on a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software (Tree Star Inc.). (9) Cell viability analysis MCF-7 cells were seeded at 5 x 10 per well in a 96-well plate. 4 Cells were seeded and left overnight. Drug-loaded liposomes (100 μg) were added to each well, and cell viability was measured at 12, 24, and 36 h using the CellVia Cell Viability Assay Kit (cat# LF-EZ1001, Abfrontier). Absorbance was detected at 440 nm using a SpectraMax Microplate Reader. Data were analyzed using SoftMaxPro software. Experiments were performed in triplicate (n=3). (10) In vivo animal studies All animal studies were approved by the Institutional Animal Care and Use Committee of the Experimental Animal Research Center of CHA University (IACUC220142). All experiments were performed in accordance with relevant guidelines and regulations. Six-week-old female BALB / c nude mice were purchased from Ja bio (Gyeonggi-do, South Korea). After 1 week, 1X10 7 MDA-MB-231 cells were injected subcutaneously into each mouse. Tumor size was 100 mm 3 When the target was reached, the mice were randomly divided into four groups (n=5 / group) and intratumoral injections of DPBS, NsLipo, LsLipo@B, or LsLipo@N (50 μl / mouse) were performed every other day (total of 7 times). Body weight and tumor volume were measured three times per week. After 28 days of treatment, the mice were sacrificed, and tumor weight and morphology were measured. To confirm tissue apoptosis, DeadEndTM Fluorometric TUNEL system kit (cat# G3250, Promega) was used. (11) Statistical analysis GraphPad Prism V.7.0 software (GraphPad Software, La Jolla, CA, USA) was used for all statistical analyses. Statistical significance was reported as follows: NS, not significant; *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001. 2. Results and Discussion (1) Changes in LED-irradiated PE / DSPE-PEG-2k liposomes Liposomes were prepared as drug delivery systems using a 15:1 molar ratio of PE to DSPE-PEG-2k. Two anticancer drugs, DOX and PTX, were then simultaneously encapsulated into the liposomes using either conventional encapsulation or LED irradiation during the liposome preparation process (Fig. 1A). While drug loading into NsLipo resulted in a slight increase in size, the sizes of blue LED-irradiated liposomes (LsLipo@B) and NIR LED-irradiated liposomes (LsLipo@N) were similar (Fig. 1B). Zeta potential (ZP) measurements were performed to determine the effect of LED (blue and NIR) irradiation on liposomes (Fig. 1C). LsLipo exhibited lower ZP values ​​than NsLipo, indicating that LED irradiation reduced the liposome surface charge and altered the liposome surface. Next, the morphology and surface structure of the liposomes were evaluated. To investigate the effect of LED irradiation on liposome morphology, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to compare NsLipo and LsLipo formulations (Fig. 1D). SEM images showed that LsLipo@B had a rougher and more uneven surface than NsLipo; on the other hand, the surface of LsLipo@N was slightly rougher. These changes affecting the liposome surface were more clearly visible in TEM images. NsLipo had a regular and smooth outer film structure, whereas LsLipo@B had a rough and irregular structure; the structure of LsLipo@N was in between. These results suggest that LED irradiation during liposome preparation induced structural modifications of the liposome membrane, which may affect the encapsulation of DOX and PTX in the liposomes. Therefore, an analysis of the drug encapsulation efficacy was performed. The liposome solutions prepared by each method were spread on glass slides, and the fluorescence intensities of DOX (red) and PTX (green) were observed using a confocal laser scanning microscope (CLSM) (Fig. 1E). Because nano-sized liposomes are difficult to observe, only micro-sized liposomes were compared. In the NsLipo sample, DOX and PTX signals were observed inside ~4-μm liposomes. Both DOX and PTX showed much higher fluorescence signals when encapsulated in LsLipo@B or LsLipo@N than in NsLipo. Next, the drug encapsulation was quantitatively analyzed (Fig. 1F). LsLipo@B and LsLipo@N were found to encapsulate approximately 114% and 70% more DOX, respectively, than that encapsulated in NsLipo. Meanwhile, LsLipo@B and LsLipo@N encapsulated approximately 26% and 23% more PTX, respectively, than that encapsulated in NsLipo. Therefore, both LsLipo@B and LsLipo@N contained higher amounts of each drug. The analysis results for each drug concentration also showed that LsLipo had a high drug loading capacity (Figure 7), confirming that liposomes can be prepared as a more efficient drug delivery system with less drug. These experiments were repeated using other model drugs to determine whether the same effects were observed when drugs other than DOX and PTX were encapsulated (Figure 8). The fluorescence intensity of FITC-dextran loaded into liposomes was 3-fold higher in LsLipo@B and 4-fold higher in LsLipo@N than in NsLipo. In addition, among liposomes loaded with TRITC, the fluorescence intensity was 1.5-fold higher in LsLipo@B and 1.6-fold higher in LsLipo@N. These results suggest that LED irradiation can be used to enhance drug encapsulation efficiency by inducing structural changes in liposomes. We evaluated the kinetics of drug release. Both NsLipo and LsLipo released 80% of the drug within the first 10 hours and then released the remainder more slowly, indicating no significant difference in drug release between NsLipo and LsLipo (Fig. 1G). (2) Evaluation of the effect of LED irradiation on liposome molecular chemical bonding To confirm the underlying mechanism of the results presented in Fig. 1, proton nuclear magnetic resonance ( 1 The chemical structures of the liposome components, PE and DSPE-PEG-2k, were analyzed after LED irradiation using H-NMR spectroscopy (Fig. 2A). 1H-NMR confirmed that LED stimulation changed the atomic composition of liposomal phospholipids (Figure 2B). The total hydrogen (H) content of phospholipids was found to be lower in LED-irradiated liposomes (i.e., blue-IR and NIR-IR subgroups, and overall, LED-IR group) than in non-irradiated liposomes (i.e., non-IR group). The number of H peaks in the range of 3.0–4.5 ppm, which is used to identify the head portion of lipids, decreased in both PE and DSPE-PEG-2k liposome components after LED irradiation. Furthermore, LED irradiation decreased the number of H peaks in the range of 0.5–2.5 ppm, which is used to identify the tail portion of lipids derived from PE and DSPE-PEG-2k. These results indicate that deprotonation occurred in both portions of each type of lipid after LED irradiation. To quantify the relative decrease in peak intensity, peak integrals were measured and compared (Fig. 9). For comparison, the integral of glycerol H (5.2 ppm) of glycerophospholipids was set to 1. A decrease in integrals (especially for PE) was observed at both 3.7 ppm and 3.2 ppm in the LED-IR group (compared to the non-IR group), indicating a decrease in the number of glycerophospholipid heads. The decrease in integrals was lower in DSPE-PEG-2k than in PE, which is due to the presence of PEG polymers in the former lipid. In addition, a decrease in integrals was observed at 1.2 ppm (representing the tail portion of the lipid) after LED irradiation. FTIR was used to investigate how LED irradiation affects various chemical bonds within lipids (Fig. 2B). The FTIR spectra showed a significant increase in the wavelength range of 2800-3000 cm in the LED-IR group (compared to the non-IR group). -1 Range (indicates the stretching frequency of the NH band) and 750-1100 cm -1A decrease in peak intensity was observed in the range (indicating the stretching frequency of the CH band). These results indicate that LED irradiation caused deprotonation of CH and NH bonds in lipids. In particular, the decrease in the stretching frequency of the NH band demonstrates that structural changes occurred in the terminal molecules of the outermost lipid layer. Based on these results, we simulated whether LED irradiation induced gap formation between lipid molecules using BIOVIA Materials Studio software (Figure 2C). The simulation confirmed that LED irradiation significantly increased the distance between PE lipid molecules. In contrast, there was no change in the distance between DSPE-PEG-2k lipids after LED irradiation, which is probably due to the presence of PEG polymers. In a mixture of PE and DSPE-PEG-2k lipids present at the same molar ratio (15:1) as the synthesized liposomes, a slight increase in the intermolecular distance was observed after LED irradiation, which was lower than the distance change observed in the PE-only group. In this experiment, “distance” refers to the separation between the nitrogen atoms of the amine head groups of the lipids, where deprotonation occurs after LED irradiation. Changes in the composition of glycerophospholipids, including the amine groups in the lipid head, can potentially affect membrane structure and function by altering the overall charge and polarity of the lipids. For example, changes in the positive charge of the amine groups can affect lipid interactions with other molecules in the membrane. Such changes in the chemical properties of the outer lipid layer can significantly affect membrane structure (e.g., changes in the density or uniformity of the lipid bilayer). The results indicate that LED irradiation altered the overall structure of the liposome membrane, particularly the hydrophilic outer layer, by altering the chemical bonds within the lipids. These changes affected how the membrane interacted with other molecules and influenced the loading and release of encapsulated drugs. (3) Evaluation of internal / external structural changes occurring in liposomes after LED irradiation using fluorescent dyes. Changes in the headgroup of PE lipids were observed after irradiation with blue light or near-infrared light. To confirm that LED irradiation actually induced these structural modifications in PE, liposomes were stained with Cy5-conjugated duramycin (a peptide that specifically binds ethanolamine) and observed (Figure 3A). Hydrophilic ethanolamine groups are located at the terminal ends of PE lipids, which are randomly arranged on the outer surface of the liposomes. It was found that duramycin interacted with ethanolamine in both LsLipo and NsLipo groups (Fig. 3B). Quantitative analysis of the strength of this interaction revealed that duramycin bound less strongly to LsLipo than to NsLipo, resulting in a significant decrease (5-fold or 10-fold) in the fluorescence intensity of duramycin bound to ethanolamine. Furthermore, duramycin interacted more weakly with LsLipo@B than with LsLipo@N. The observed decrease in duramycin intensity in the LsLipo group (compared to NsLipo) may be due to the modification of the chemical structure of ethanolamine by LED irradiation. Both blue and NIR wavelengths have shown the potential to modify the chemical structure of ethanolamine. Similar results were obtained when duramycin-labeled liposomes were observed using CLSM (Fig. 3C). Line profiling in the magnified CLSM images of the liposomes revealed that the intensity and distribution of duramycin in LsLipo@B and LsLipo@N were approximately four times lower than in NsLipo (Fig. 10). Therefore, LED stimulation of liposomes was confirmed to induce a structural modification of ethanolamine. The surface charges of duramycin and liposomes, as well as the change in surface charge after binding, were measured (Fig. 11). Duramycin has a surface charge of approximately -6 mV, whereas liposomes have a surface charge of approximately 5 mV (LsLipo has a lower surface charge than NsLipo; LsLipo@B: 1.5 mV, LsLipo@N: 0.5 mV). When liposomes were labeled with duramycin, the surface charge of NsLipo was significantly reduced (to -3 mV). However, duramycin labeling of LsLipo only slightly reduced its surface charge. These findings suggest that the surface charge of the liposomes was altered by denaturation of the outermost ethanolamine moiety. Furthermore, the negative charge of duramycin did not affect the surface charge of the liposomes, indicating that duramycin could not effectively bind to liposomes after LED irradiation. These results highlight the potential use of LED irradiation as a tool for modifying the properties of liposomes and other biological membranes. Liposomes were labeled with the lipophilic fluorescent dyes DiI (red) and DiO (green) to analyze changes in the phospholipid tails (Fig. 3D). Phospholipid tails have hydrophobic properties and thus interact with the lipophilic dyes. Changes in dye binding can be assessed by comparing fluorescence intensities under different conditions. Using this assay, LsLipo@B and LsLipo@N were found to produce red and green signals of similar intensity to NsLipo. Therefore, it was confirmed that LED irradiation did not significantly affect the phospholipid tails of the liposomes. (4) Measurement of whether LED irradiation changes liposome stiffness To understand how changes in the chemical composition of lipids affect the liposome membrane structure and mechanical properties, the effect of LED irradiation on the physical properties of liposomes was investigated. First, structural changes in empty liposomes (unloaded with drugs) were observed by measuring the shape and height using atomic force microscopy (AFM) (Figure 4A). NsLipo was found to be larger and more rigid than LsLipo (both LsLipo@B and LsLipo@N). AFM measures attractive and repulsive forces to determine the shape of a sample based on the strength of the interaction between the atoms on the cantilever and the sample surface (e.g., K. Iwamoto, et al., Biophys J 2007,93, 1608). Exposure of phospholipids to LED irradiation induced deprotonation (Figure 12), and LsLipo@B and LsLipo@N, composed of deprotonated lipids, were found to be less rigid than NsLipo. Overall, these findings suggest that changes in the chemical structure of liposomes after LED irradiation significantly affect the membrane structure and physical properties of liposomes. To investigate the effect of LED irradiation on the mechanical properties of liposomes, Young's modulus, a measure of stiffness, was measured (Fig. 4B). As a result, NsLipo (680 MPa) exhibited a higher Young's modulus than LsLipo (380–400 MPa), indicating that NsLipo possesses a denser membrane structure that better withstands external pressure. Therefore, chemical and structural changes induced by LED irradiation lead to membrane deformation, reducing the Young's modulus of liposomes and thus their resistance to external stress. The effect of LED irradiation on drug-loaded liposomes was investigated using AFM (Fig. 4C). Similar to the results obtained using empty liposomes, LED irradiation significantly altered the physical properties of drug-loaded liposomes. For example, LsLipo@B and LsLipo@N were found to have lower rigidity than NsLipo. These results help explain why LsLipo exhibits higher drug encapsulation efficiency than NsLipo. Mechanistically, LED irradiation alters the structure of the phospholipid head groups constituting the outer layer of LsLipo (chemical change), which subsequently reduces liposome rigidity (physical change), providing favorable conditions for drug encapsulation. Furthermore, the increased liposome flexibility after LED irradiation likely enhanced its ability to enter target cells. (5) Comparison of anticancer effects of NsLipo and LsLipo in MCF7 cell lines The anticancer efficacy of drug-loaded liposomes was evaluated in cancer cells. MCF7 cells (a breast cancer cell line) were treated with NsLipo and LsLipo containing PTX and DOX, and images were taken at 2-hour intervals for up to 12 hours (Fig. 13). After 4 hours of treatment, similar small amounts of fluorescence were observed in cells treated with LsLipo or NsLipo. Notably, after 8 hours, the amount of PTX (green) in the cytoplasm of cancer cells treated with LsLipo was higher than that in cells treated with NsLipo. After 12 hours, a higher amount of DOX (red) was observed in the nucleus of cancer cells treated with LsLipo than in cells treated with NsLipo. These results indicate that LsLipo encapsulates greater amounts of PTX (green) and DOX (red) than NsLipo, enabling efficient delivery to the cytoplasm or nucleus of cancer cells. To quantify the drug delivery efficiency of each liposome formulation, the intracellular amounts of each drug in liposome-treated cancer cells were measured every 2 hours for 12 hours using a flow cytometer (Fig. 14). Significantly higher amounts of DOX and PTX were found in cancer cells treated with LsLipo than in those treated with NsLipo (Fig. 5B). These results demonstrate that LED irradiation increased the amount of drug loaded into liposomes, which was then effectively delivered to cancer cells. The anticancer activity of each liposome formulation was measured every 2 hours for 12 hours, and then compared to whether LED-irradiated liposomes killed cancer cells more effectively than non-irradiated liposomes (Fig. 15). After 12 hours of treatment (Fig. 5C), 15% of cells in the untreated (NT) group reached late apoptosis, while 48% of cells in the NsLipo group reached late apoptosis due to drug-induced death. However, LsLipo@B and LsLipo@N induced 73.7% and 77.74% of MCF7 cells, respectively, demonstrating greater apoptosis than that induced by NsLipo. These data suggest that LsLipo contained more DOX and PTX than NsLipo, thus inducing a higher level of apoptosis. To assess the retention of PTX and DOX in cancer cells, intracellular drug levels were detected 24 and 48 h after liposome treatment (Fig. 5D). At 24 h after LsLipo treatment, DOX was primarily detected in the nucleus, whereas PTX was detected in the cytoplasm. Both drugs were detected in high amounts in the cells 48 h after liposome treatment. Considering the anticancer activities of DOX and PTX, which inhibit DNA replication and tubulin assembly, respectively, the retention times in the cytoplasm and nucleus are directly proportional to their anticancer efficacy. By quantifying the fluorescence signals generated by the drugs, we confirmed that LsLipo delivered higher amounts of PTX and DOX to cells than NsLipo, and that both drugs were retained intracellularly at both 24 and 48 h after treatment (Fig. 5E). The viability of MCF7 cells was measured every 12 hours for 48 hours (Fig. 5F). The viability of cancer cells treated with LsLipo was significantly lower than that of cancer cells treated with NsLipo. These results suggest that LsLipo delivered a higher amount of drug to cancer cells, resulting in a higher level of cancer cell death. (6) In vivo antitumor efficacy of LsLipo loaded with anticancer drug cocktail The antitumor efficacy of LED-irradiated and non-irradiated liposomes loaded with DOX and PTX was evaluated in a xenograft mouse model of triple-negative breast cancer. LsLipo or NsLipo formulations were injected into tumors of nude mice every other day for a total of seven times (Fig. 6A). Tumors were observed 28 days after liposome administration, and tumors in LsLipo-treated mice were found to be smaller than those in NsLipo-treated mice (Fig. 6B). The most significant decrease in tumor growth rate was observed in mice treated with LsLipo@N (compared to mice treated with conventional liposomes) (Fig. 6C). These results demonstrate that LsLipo@N contains higher amounts of DOX and PTX than blue light-irradiated or non-irradiated liposomes, resulting in a more potent in vivo anticancer effect. Tumor weights examined 28 days after treatment revealed that mice treated with LsLipo@N developed significantly smaller tumors than those treated with the other two liposome formulations (Figs. 6D-6E). There was no significant difference in body weight between the groups, indicating that the liposomes were non-toxic and did not cause significant side effects in the mice (Fig. 6F). Twenty-eight days after liposome treatment, tumor tissues from the mice were examined using TUNEL and hematoxylin and eosin (H&E) staining (Fig. 6G). H&E staining analysis showed that tumor tissues from control mice had a dense structure, whereas those from liposome-treated mice had a more porous appearance due to increased tumor cell death. The LsLipo group exhibited a larger area of ​​tumor tissue exfoliation than the NsLipo group. The LsLipo@N group exhibited the greatest amount of tumor tissue eradication. TUNEL analysis is a unique tool used to visualize and quantify apoptosis in tumor tissues. A stronger TUNEL signal was observed in the LsLipo group than in the NsLipo group. Specifically, the LsLipo@N formulation demonstrated the most effective drug delivery, exhibiting the highest intensity of green fluorescence. These findings provide a rationale for the use of drug delivery particles, such as liposomes, to achieve sustained drug release from tumors, and also for the manipulation of particles using LED light, particularly light at NIR wavelengths, for effective tumor eradication. 3. Conclusion This study presents a novel method for encapsulating insoluble drugs into liposomes by inducing structural changes in the liposome membrane using LED irradiation. It was found that large amounts of anticancer drugs, such as DOX and PTX, were encapsulated into liposomes, enabling efficient delivery to tumor tissues. Mechanistically, these results demonstrate that LED irradiation alters the lipid chemistry of the liposome surface, which in turn alters the physical properties of the liposomes, facilitating the encapsulation of poorly soluble drugs. Furthermore, LED-irradiated liposomes effectively reduced tumor size and weight in a breast cancer mouse model. Furthermore, the inventors demonstrated that the LED irradiation method for drug encapsulation is applicable to drugs other than DOX and PTX. Overall, these results suggest that LED-irradiated liposomes have significant potential for use in cancer therapy and regenerative medicine, as well as in other drug delivery applications.

Claims

A method for producing liposomes encapsulating an anticancer agent, comprising: (a) a step of irradiating liposomes with blue light or near-infrared light; and (b) a step of encapsulating an anticancer agent in the light-irradiated liposomes obtained in step (a).

2. A manufacturing method according to claim 1, characterized in that the liposome is formed from a mixture of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000].

3. A manufacturing method according to claim 2, characterized in that the molar ratio of 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] in the mixture is 10 to 25:

1.

4. A manufacturing method according to claim 1, characterized in that the blue light has a wavelength of 400 to 495 nm.

5. A manufacturing method according to claim 1, characterized in that the near-infrared light has a wavelength of 780 to 2,500 nm.

6. A manufacturing method according to claim 1, characterized in that the light irradiation is performed for 0.5 to 2 hours.

7. A manufacturing method according to claim 1, characterized in that the anticancer agent is doxorubicin or a pharmaceutically acceptable salt thereof; paclitaxel or a pharmaceutically acceptable salt thereof; or a combination of doxorubicin or a pharmaceutically acceptable salt thereof and paclitaxel or a pharmaceutically acceptable salt thereof.

8. A pharmaceutical composition for treating cancer, comprising, as an active ingredient, a liposome encapsulating an anticancer agent obtained by a manufacturing method according to any one of claims 1 to 7.

9. A pharmaceutical composition according to claim 8, characterized in that the cancer is breast cancer, non-small cell lung cancer, or skin cancer.

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